The effects of simulated wastewater nutrient amendments on ... MSc. Thesis...and make accessible my...

The effects of simulated wastewater nutrient amendments on Sphagnum productivity and decomposition within a subarctic ribbed fen1

by

Amanda Lavallee

Thesis submitted in partial fulfillment of the requirements for the degree of Master of Science (MSc.) in Biology

The Faculty of Graduate Studies Laurentian University

Sudbury, ON

ãAmanda Lavallee, 2017

ii

THESIS DEFENCE COMMITTEE/COMITÉ DE SOUTENANCE DE THÈSE

Laurentian Université/Université Laurentienne

Faculty of Graduate Studies/Faculté des études supérieures

Title of Thesis

Titre de la thèse The effects of simulated wastewater nutrient amendments on Sphagnum productivity

and decomposition within a subarctic ribbed fen¹

Name of Candidate

Nom du candidat Lavallée, Amanda

Degree

Diplôme Master of Science

Department/Program Date of Defence

Département/Programme Biology Date de la soutenance July 21, 2017

APPROVED/APPROUVÉ

Thesis Examiners/Examinateurs de thèse:

Dr. Daniel Campbell

(Supervisor/Directeur de thèse)

Dr. Nathan Basiliko

(Committee member/Membre du comité)

Dr. Graeme Spiers

(Committee member/Membre du comité)

Approved for the Faculty of Graduate Studies

Approuvé pour la Faculté des études supérieures

Dr. David Lesbarrères

Monsieur David Lesbarrères

Dr. Tim Moore Dean, Faculty of Graduate Studies

(External Examiner/Examinateur externe) Doyen, Faculté des études supérieures

ACCESSIBILITY CLAUSE AND PERMISSION TO USE

I, Amanda Lavallée, hereby grant to Laurentian University and/or its agents the non-exclusive license to archive

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duration of my copyright ownership. I retain all other ownership rights to the copyright of the thesis, dissertation or

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dissertation, or project report. I further agree that permission for copying of this thesis in any manner, in whole or in

part, for scholarly purposes may be granted by the professor or professors who supervised my thesis work or, in their

absence, by the Head of the Department in which my thesis work was done. It is understood that any copying or

publication or use of this thesis or parts thereof for financial gain shall not be allowed without my written

permission. It is also understood that this copy is being made available in this form by the authority of the copyright

owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted

by the copyright laws without written authority from the copyright owner.

III

Abstract

Peatlands dominate the flat landscape of the Hudson Bay Lowland (HBL). Sphagnum mosses are

the key peat-generating plants allowing for important ecosystem services such as carbon storage,

climate regulation, and water polishing. The HBL is a location for current and proposed

industrial mining development projects, and its peatlands may become increasingly used to

polish treated wastewater from mining camps. This study focuses on biological changes in the

Sphagnum moss community associated with the addition of simulated treated domestic

wastewater to a subarctic ribbed fen (a wetland type commonly found throughout the HBL). We

determined how the nutrient additions affected the productivity, decomposition, and nutrient

ratios, within the ponds and raised peatland ridge components of the ribbed fen. Field experiment

results show between a four to twelvefold increase in productivity rates of the low-lying

Sphagnum rubellum species, and a twofold increase in productivity for the higher hummock or

ridge dominating species Sphagnum fuscum in locations closest to the point source of nutrient

effluent. Regions of the experimental ribbed fen greater than 50 m away from the point source

showed little difference in productivity rates, nutrient content, or decomposition rate than the

reference fen levels. No significant changes to the rate of decomposition of Sphagnum were

observed with relation to distance away from point source nutrients as the experimental fen

decomposition rates were comparable to the reference fen rates. A laboratory peat incubation

experiment was conducted to determine how increasing exposure to the wastewater nutrients

would affect Sphagnum decay potentials. Lab results indicate that greater concentrations of

nutrient additions to incubation environments did not significantly increase the amount of CO2 or

CH4 emissions. However, origin of the peat and the species of Sphagnum moss comprising the

peat was found to be important factors contributing to Sphagnum decomposability and

greenhouse gas emissions. Peat formed within a nutrient enriched location produced significantly

greater CO2 and CH4 emissions than peat originating from non-fertilized locations, and hollow

dominant Sphagnum species show greater decomposability than hummock forming species.

Therefore, this study suggests that in the short-term subarctic peatlands exposed to nutrient levels

comparable to that present in treated domestic wastewater will increase their capacity to generate

Sphagnum-peat and store carbon. This experimental research aids in understanding to what

degree plants mediate shifts in ecosystem dynamics within subarctic ribbed fens. Both policy

IV

makers and industries will consult these results for mining development projects within the HBL

and elsewhere in subarctic and boreal biomes.

Keywords: Ribbed fen, subarctic peatland, Hudson Bay Lowlands, Sphagnum moss, carbon

storage, treatment wetlands

V

Acknowledgements

Many people have supported and assisted me in the completion of this Masters research project

and deserve my sincerest thanks. Without their support over these past two years I could not

have gotten here.

First, I would like to express my utmost gratitude to my academic supervisor Dr. Daniel

Campbell. His continued advice, direction, and encouragement through this process shows he

truly cares about his student’s success and well-being, and for that I hold him in the highest

respect. I am honoured and lucky to have worked with such a great mentor. I am also very

thankful for my advisory committee, Dr. Basiliko and Dr. Spiers, whose knowledge and advice

were greatly appreciated and very helpful.

I am grateful for my fellow collaborators within the Canadian Network for Aquatic

Ecosystem Services (CNAES). Special thanks to Dr. Colin McCarter and Dr. Jonathan Price for

their design and execution of the experimental fen project. Thank you to Dr. Brian Branfireun,

Lauren Twible, Nicole Balliston, and Dr. John Gunn for their support and collaboration

throughout this research. Further thanks to Angela Borynec and Ainsley Davison for their

assistance in the field, as well as Andrea Hanson and Brittany Rantala-Sykes, as all of you

provided me support, encouragement, and excellent companionship.

Special thanks go out to all De Beers Victor Mine employees who contributed to the

success of this research. Thank you Brian Steinback, Stephen Monninger, Terry Ternes, Rod

Blake, and Anne Bouche for their onsite management, coordination of field logistics, and

administration work. Thank you to all members of the Victor Mine Environment and

Reclamation departments, and special thanks to Tara Despault, Aline De Chevigny for assistance

in the lab, as well as Nick Gagnon, Jake Carter, Jordan Edwards, and Isaiah Hollinger for their

transportation to research sites throughout the past two years of fieldwork. All Victor Mine

employees were a pleasure to get to know, very welcoming, and made time onsite a positive and

memorable experience.

Thank you to fellow Vale Living with Lakes graduate students and professors whom have

either provided me with training in the lab, advice on data analysis, or allowed me to use their

equipment, specifically Michael Carson, Gretchen Lescord and Dr. Tom Johnston.

VI

I would like to thank all funders of this research including the Natural Sciences and

Engineering Research Council of Canada (NSERC), the NSERC Canadian Network for Aquatic

Ecosystem Services (CNAES), De Beers Canada and the Northern Scientific Training Program.

Lastly, I would like to thank my friends and family for their unconditional love and

support. Thank you for encouraging me with my love and fascination for the natural sciences and

environment, for being patient with me, and for listening to me rant and rave about Sphagnum.

VII

Table of Contents

Preface ............................................................................................................................................ 1

Literature Cited ........................................................................................................................... 3

CHAPTER 1: The effects of simulated treated domestic wastewater on Sphagnum

productivity, decomposition, and nutrient dynamics in a subarctic ribbed fen ..................... 5

Abstract ....................................................................................................................................... 6

Introduction ................................................................................................................................. 8

Methods..................................................................................................................................... 12

Results ....................................................................................................................................... 17

Discussion ................................................................................................................................. 20

Conclusion ................................................................................................................................ 25

Acknowledgements ................................................................................................................... 26

Literature Cited ......................................................................................................................... 27

CHAPTER 2: Decomposition of Sphagnum peat from a ribbed fen receiving simulated

treated wastewater: an incubation experiment along a nutrient loading gradient .............. 44

Abstract ..................................................................................................................................... 45

Introduction ............................................................................................................................... 47

Methods..................................................................................................................................... 50

Results ....................................................................................................................................... 53

Discussion ................................................................................................................................. 56

Conclusion ................................................................................................................................ 65

Acknowledgements ................................................................................................................... 66


Research Implications ................................................................................................................ 78


Appendix ...................................................................................................................................... 84

VIII

List of Tables

CHAPTER 1

Table 1. Spearman’s correlation coefficients among Sphagnum productivity, decomposition, first principal component (PC1) of the nutrient data and the C:N ratio within Sphagnum collected from the experimental fen. S. fuscum is shown in the upper triangular matrix (grey) and S. rubellum in the lower triangular matrix (white). Correlation coefficients in bold are significant at P < 0.05 based on a two-tailed test. ……………………………….32

Table 2. Analyses of variance for productivity and decomposition separated by species, as a

function of the fen site (experimental or reference), distance downgradient and their interaction. Results with type I error < 10% are bolded. ………………………………….33

Table 3. Correlation (loadings) between the original variables and the first two principal

components from the principal component analysis (PCA). ……………………………...34 Table 4. Analyses of variance for the first principal component of plant nutrient content and for

the C:N ratio, separated by species, as a function of fen site (experimental or reference), distance downgradient, and their interaction. Results with type I error < 10% are bolded. ……………………………………………………………………………………………..35

CHAPTER 2 Table 1. Separate analyses of variance by Sphagnum species of 40 day anaerobic CO2 release, 24

hour aerobic CO2 release and 40 day anaerobic CH4 release during the incubation experiment as a function of Sphagnum origin, nutrient amendment and their interaction. Analyses were based on log-transformed data. Results with type I error level < 5% are shown in bold. …………………………………………………………………………….73

IX

List of Figures

CHAPTER 1

Figure 1. Google Earth satellite image of the De Beers Victor Mine site (June 2013), showing the experimental fen and the reference fen. The experimental fen is 8.5 km northwest from the reference fen. ……………………………………………………………………………...36

Figure 2. Box plots of monthly temperature of Pond 1(white) and Ridge 1 (grey) at 10 cm depth

within the experimental fen during the 2016 growing season. ……………………………37 Figure 3. The productivity of Sphagnum fuscum (left) and Sphagnum rubellum (right) over a six-

week period in the summer of 2015 (top) and the summer of 2016 (bottom). Direction of hydrological flow runs from top to bottom within each fen, following the sequence of pool numbers. The arrow on Pool 1 of the experimental fen marks the location of the point source of nutrient addition. The reference fen is 8.5 km away from the experimental fen, but they are shown together here. ………………………………………………………..38

Figure 4. Productivity over 6 weeks during the growing season in 2015 (top) versus 2016

(bottom) for Sphagnum fuscum (left) and Sphagnum rubellum (right) as a function of distance from the discharge point in the experimental fen and the top edge of the fen in the reference fen. Solid lines and solid circles represent experimental fen data, and open circles and dashed lines represent the reference fen data. The vertical dotted line marks the point source input of nutrients in the experimental fen. Samples with negative distance values are located upgradient from the nutrient discharge point. …………………………………….39

Figure 5. The productivity of Sphagnum fuscum (top left) and Sphagnum rubellum (top right)

over a 12-week period in the summer of 2016, and the decomposition rate of S. fuscum (bottom left) and S. rubellum (bottom right) over one year from July 2015 to July 2016. Direction of hydrological flow runs from top to bottom within each fen, following the sequence of pool numbers. The arrow on Pool 1 of the experimental fen marks the discharge point source for nutrient addition. The reference fen is 8.5 km away from the experimental fen, but they are shown together here. ……………………………………...40

Figure 6. Linear regression plots of the productivity over 12 weeks during the growing season in

2016 (top) and annual decomposition (bottom) for Sphagnum fuscum (left) and Sphagnum rubellum (right) in the experimental fen (solid circles and lines) and the reference fen (open circles and dashed lines). The vertical dotted line marks the location of the discharge point of nutrients, and negative distance values correspond to sample sites up gradient from the point source. …………………………………………………………………………...41

Figure 7. Principal component ordination plot of the nutrient content of all three Sphagnum

species in the experimental fen (solid) and reference fen (open). The percent variation explained by principal component is shown in parentheses on each axis. Together they summarize 91% of the total variation of the nutrient content in the Sphagnum species. ……………………………………………………………………………………………..42

Figure 8. Linear regression plots of (A) the first principal component of Sphagnum nutrient

content (PC1; left) and (B) the C:N ratio (right) in the experimental fen (solid circles and lines) and the reference fen (open circles and dashed lines). The vertical dotted line marks the location of the discharge point of nutrients, and negative distance values correspond to sample sites up gradient from the point source. …………………………………………..43

CHAPTER 2 Figure 1. Box plots of the (A) anaerobic 40 day CO2 production, (B) 24 hour aerobic CO2

production, and (C) the 40 day anaerobic CH4 production during the incubation of peat from Sphagnum fuscum (brown), S. rubellum (red) and S. majus (green) collected at the start and the end of the experimental fen and at the reference fen, across all nutrient amendment treatments. Letters above box plots show results of post-hoc Tukey test with a 5%Type I error rate, plots with no letters indicates no significant differences across peat origin. ……………………………………………………………………………………..74

X

Figure 2. Box plots of the (A) anaerobic 40 day CO2 production, (B) 24 hour aerobic CO2

production, and (C) the 40 day anaerobic CH4 production during the incubation of peat from Sphagnum fuscum (brown), S. rubellum (red) and S. majus (green) as a function of nutrient amendment levels ranging from zero (no amendment) to up to 10x the field rate of nutrient amendment. Letters above box plots show results of post-hoc Tukey test with a 5%Type I error rate, plots with no letters indicates no significant differences across nutrient amendment ……………………………………………………………………….75

Figure 3. Box plots of the CO2 produced after 24 hours under aerobic conditions for S. fuscum at

each nutrient amendment level, ranging from zero (no amendment) to up to 10x the field rate of nutrient amendment, at all three peat origins. Letters above box plots refer to similar groups based on post hoc Tukey test with a 5%Type I error rate. ………………..76

Figure 4. Box plots of the CH4 produced after 40 days under anaerobic conditions for S. rubellum

at each nutrient amendment level, ranging from zero (no amendment) to up to 10x the field rate of nutrient amendment, at all three peat origins. Letters above box plots refer to similar groups based on post hoc Tukey test with a 5%Type I error rate. ………………..77

XI

List of Appendix Figures

Figure A1. Scatter plot of total nitrogen content (percent mass) within Sphagnum fuscum (top), Sphagnum rubellum (centre), and Sphagnum majus (bottom) over distance (m), experimental fen (solid black circles) and the reference fen (open circles). The vertical dashed line marks the location of the discharge point of nutrients, and negative distance values correspond to sample sites up gradient from the point source……………………..78

Figure 2A. Scatter plot of total sulfur content (percent mass) within Sphagnum fuscum (top),

Sphagnum rubellum (centre), and Sphagnum majus (bottom) over distance (m), experimental fen (solid black circles) and the reference fen (open circles). The vertical dashed line marks the location of the discharge point of nutrients, and negative distance values correspond to sample sites up gradient from the point source……………………..79

Figure 3A. Scatter plot of phosphorus concentration (mg kg-1) within Sphagnum fuscum (top),


Figure 4A. Scatter plot of potassium concentration (mg kg-1) within Sphagnum fuscum (top),


Figure 5A. Scatter plot of calcium concentration (mg kg-1) within Sphagnum fuscum (top),


Figure 6A. Scatter plot of magnesium concentration (mg kg-1) within Sphagnum fuscum (top),


1

Preface

Peatlands store large amounts of terrestrial carbon because the rate of primary productivity

exceeds the rate of organic matter decay. They cover approximately 3% of the Earth’s land area

and store 15 to 30 % of global soil carbon, especially in boreal and subarctic landscapes

(Limpens et al. 2008; Yu et al. 2010). These northern peatlands include three of the ten largest

wetlands in the world (Keddy et al. 2009). The Hudson Bay Lowland (HBL) is the third largest

in area, spanning 373,700 km2 across north-central Canada, but it is perhaps the most carbon-

dense (Abraham and Keddy 2005; Gorham 2003). The key peat-forming vegetation contributing

to the accumulation of peat on the landscape is Sphagnum moss (Abraham and Keddy 2005),

which has been identified as the keystone genus of the north because of its inherent resistance to

decay and its survival in cool climate, nutrient poor conditions (Rochefort 2000). Current and

projected changes in northern climate, especially around Hudson and James Bays (McLaughlin

and Webster 2013) place an increased spotlight on this carbon sequestration service of the

peatlands of the HBL, especially the contribution of Sphagnum.

Peatlands provide an array of regulating ecosystem services in addition to the sequestration

and storing carbon, such as erosion protection, water quantity regulation and water filtration and

purification (Kimmel and Mander 2010). Humans have commonly used the filtering function of

wetlands ecosystems such as marshes to polish secondarily-treated domestic wastewater (Kadlec

and Wallace 2009), but we have less often harnessed northern peatlands to polish wastewater

(Kadlec 2009; Ronkanen and Klove 2009). This wetland polishing is a tertiary treatment which

is meant to remove removes excess nutrients and other contaminants.

The HBL peatlands are currently considered pristine due to their lack of large-scale human

or industrial developments, however, large deposits of mineral resources have been discovered in

2

the area and an increase in industrial and human developments may arise within the coming

decades (Far North Science Advisory Panel 2010). The increasing numbers of remote mining

camp operations, as well as an increasing population within indigenous communities would

result in a greater need for the HBL peatlands to perform wastewater polishing services. Few

studies have assessed the ability of Canadian subarctic peatlands to polish treated domestic

wastewater. There is a lack of understanding on the biological and hydrological response of

peatlands within northern subarctic climates, such as the HBL, to cope with high nutrient loading

at a point source resulting from anthropogenic developments.

In a collaborative study, McCarter and Price (2017), and McCarter et al. (2017) provided

insight on the hydrological response and nutrient transport, while Twible and Branfireun (in

preparation) provide insight on the biogeochemical response of a subarctic ribbed fen receiving

simulated secondarily-treated domestic wastewater from a remote mining camp operation. This

current thesis aims to complement the previous and on-going research by providing insight into

the biological response to the point source nutrient loading by assessing the impact on the rates

of productivity and decomposition of key Sphagnum species and the nutrient content and C:N

ratios within Sphagnum tissues.

My thesis is composed of two research chapters, each written in manuscript form. Chapter

1 focuses on (i) measuring the field rates of Sphagnum productivity and decomposition, as well

as (ii) determining nutrient concentrations within the Sphagnum tissues themselves across the

hydrological and nutrient gradient of the experimental fen, as compared to a non-fertilized

reference fen. Chapter 2 is a laboratory peat incubation experiment that focuses on determining

the microbial decomposition (CO2 and CH4 emissions) of three species of Sphagnum mosses

exposed to increasing concentrations of nutrient loading. Both chapters work towards

3

understanding how nutrient additions associated with treated domestic wastewater would impact

Sphagnum-peat formation and decomposition. Shifts to the rates of Sphagnum productivity and

decay would directly impact the quantity and quality of carbon storage, the hydrology and the

wastewater polishing services provides by the HBL peatlands.

I am submitting my Master of Science thesis in the form of two manuscripts. Chapter 1 is a

manuscript co-authored by my thesis supervisor, Daniel Campbell, Jim McLaughlin from the

Ontario Forestry Research Institute and myself. Chapter 2 is a manuscript co-authored by Daniel

Campbell and myself. For both chapters, I assisted in planning the experiments along with

Daniel Campbell, and I was responsible for conducting these experiments in the field and in the

lab. I was in charge of sampling, data analysis, and writing. Daniel Campbell contributed to the

experimental design and planning of both experiments, and helped determine which parameters

to focus on and was also involved with brainstorming the statistical analysis and editing my

writing. Given my role in these manuscripts, I am submitting them as my Master of Science

thesis.

Literature Cited

Abraham, K.F. & Keddy C.J. (2005). The Hudson Bay Lowland. In L.H. Fraser and P.A. Keddy (eds.). The world’s largest wetlands: Ecology and conservation. Cambridge, UK: Cambridge University Press. pp 118-148.

Far North Science Advisory Panel. (2010). Science for a Changing Far North. The Report of the

Far North Science Advisory Panel. A report submitted to the Ontario Ministry of Natural Resources.

Gorham, E., Janssens, J., & Glaser, P. (2003). Rates of peat accumulation during the postglacial

period in 32 states from Alaska to Newfoundland, with special emphasis on northern Minnesota. Canadian Journal of Botany, 81, 429-438.

4

Kadlec, R. H., & Wallace, S. D. (2009). Treatment Wetlands (2nd ed.). Boca Raton, FL: CRC Press, Taylor & Francis Group.

Kadlec, R. H. (2009). Wastewater treatment at the Houghton Lake wetland: Hydrology and

water quality. Ecological Engineering, 35, 1287–1311. doi:10.1016/j.ecoleng.2008.10.001 Keddy, P. A., L. H. Fraser, A. I. Solomeshch, W. J. Junk, D. R. Campbell, M. T. K. Arroyo, &

C. J. R. Alho. (2009). Wet and wonderful: the world's largest wetlands are conservation priorities, BioScience, 59(1), 39-51.

Kimmel, K., & Mander, Ü. (2010). Ecosystem services of peatlands: Implications for

restoration. Progress in Physical Geography, 34(4), 491-514. Limpens, J., Berendse, F., Blodau, C., Canadell, J. G., Freeman, C., Holden, J., ... & Schaepman-

Strub, G. (2008). Peatlands and the carbon cycle: from local processes to global implications–a synthesis. Biogeosciences, 5(5), 1475-1491.

McCarter, C. P. R., Branfireun, B. A., & Price, J. S. (2017). Nutrient and mercury transport in a

sub-arctic ladder fen peatland subjected to simulated wastewater discharges. Science of The Total Environment, 609, 1349-1360.

McCarter, C. P., & Price, J. S. (2017). The transport dynamics of chloride and sodium in a ladder

fen during a continuous wastewater polishing experiment. Journal of Hydrology, 549, 558-570.

McLaughlin, J., & Webster, K. (2013). Effects of a changing climate on peatlands in permafrost

zones: a literature review and application to Ontario's Far North (No. CCRR-34). Ontario Forest Research Institute.

Rochefort, L. (2000). Sphagnum—a keystone genus in habitat restoration. The

Bryologist, 103(3), 503-508. Ronkanen, A.-K., and Klove, B. 2009. Long-term phosphorus and nitrogen removal processes

and preferential flow paths in northern constructed peatlands. Ecological Engineering, 35(5), 843-855 doi:10.1016/j.ecoleng.2008.12.007.

Yu, Z., J. Loisel, D. P. Brosseau, D. W. Beilman, & S. J. Hunt. (2010). Global peatland

dynamics since the Last Glacial Maximum. Geophys. Res. Lett., 37, L13402, doi:10.1029/2010GL043584.

5

CHAPTER 1: The effects of simulated treated domestic wastewater on Sphagnum productivity, decomposition, and nutrient dynamics in a subarctic ribbed fen

Amanda Lavallee1,2, Jim McLaughlin3 and Daniel Campbell1,4

1 Vale Living with Lakes Centre, Laurentian University, Sudbury, Ontario, Canada P3E 2C6

2 Department of Biology, Laurentian University, Sudbury, Ontario, Canada P3E 2C6

3 Ontario Forestry Research Institute, Sault Ste. Marie, Ontario, Canada P6A 2E5

4 School of the Environment Laurentian University, Sudbury, Ontario, Canada P3E 2C6

6

Abstract

Peatlands dominate the flat landscape of the Hudson Bay Lowland (HBL). Sphagnum mosses are

the key peat-generating plants allowing for important ecosystem services such as carbon storage

and water polishing. The HBL also has current and proposed industrial mining development

projects, and its peatlands may become increasingly used to polish secondarily-treated

wastewater from mining camps. We examined biological changes in the plant community

associated with the addition of simulated secondarily-treated wastewater to a subarctic ribbed

fen, a wetland type commonly found throughout the HBL. We determined how the nutrient

additions affected the productivity, decomposition, and nutrient ratios within the ponds and

raised peatland ridge components of the ribbed fen. Our results show between a four to

twelvefold increase in productivity rates of the low-lying Sphagnum rubellum species, and a

twofold increase in productivity for the higher hummock or ridge-dominating species Sphagnum

fuscum in locations closest to the point source of nutrient effluent. Regions of the experimental

ribbed fen greater than 50 m away from the point source showed little difference in productivity

rates or nutrient content than the reference fen levels. No significant changes to the rate of

decomposition of Sphagnum were observed with relation to distance away from point source

nutrients as the experimental fen decomposition rates were comparable to the reference fen rates.

Sphagnum productivity per year remained greater than mass lost to decomposition. Therefore,

this study suggests that, in the short-term, subarctic peatlands exposed to nutrient levels

comparable to that present in treated domestic wastewater will increase their capacity to generate

Sphagnum-peat and store carbon. This experimental research aids in understanding to what

degree plants mediate shifts in ecosystem dynamics within subarctic ribbed fens. Policy makers,

7

community planners, and industries may consult these results for mining development projects

within the HBL and elsewhere in subarctic and boreal biomes.

Keywords: Subarctic peatland, ridge-pool sequence, Hudson Bay Lowland, Sphagnum moss,

nutrient enrichment, tertiary treatment, wastewater polishing, remote resource development,

mining

Data repository: Scholars Portal Dataverse

8

Introduction

Peatlands have a rate of primary production which exceeds their rate of decomposition, so they

sequester and store carbon as peat. They cover approximately 3% of the Earth’s land area and

store 15 to 30 % of global soil carbon, especially in boreal and subarctic landscapes (Limpens et

al., 2008; Yu et al., 2010). These northern peatlands remain largely pristine (Potapov et al.,

2008; Lee et al., 2010) and include three of the ten largest wetlands in the world (Keddy et al.,

2009). The Hudson Bay Lowland (HBL) is the third largest wetland in area, spanning 373,700

km2 across north-central Canada, but it is perhaps the most carbon-dense (Gorham, 1991;

Abraham and Keddy, 2005). Peatlands provide an array of regulating ecosystem services in

addition to the sequestration and storing carbon, such as erosion protection, water quantity

regulation and water filtration and purification (Kimmel and Mander, 2010). Humans have

commonly used the filtering function of wetlands ecosystems such as marshes to polish

secondarily-treated domestic wastewaters (Kadlec and Wallace, 2009), but we have less often

harnessed northern peatlands to polish wastewater (Kadlec, 2009; Ronkanen and Klove, 2009,

McCarter et al., 2017).

Northern peatlands are dominated by ombrotrophic bogs and ribbed fens (Keddy et al.,

2009; Riley, 2011). Bogs only receive water from precipitation and serve as water storage

complexes, whereas fens act as conveyors of water from peatlands across the landscape during

periods of high hydrological connectivity (Quinton et al., 2003). Ribbed fens have a repeating

pattern of pool-ridge-pool morphology with slow hydrological flow running perpendicular to the

raised peat ridges (Price and Maloney, 1994; Quinton and Roulet, 1998). Hydrological

connectivity within a ribbed-fen occurs by a spill-and-fill mechanism when the water table rises

above the height of the peat ridge (Spence and Woo, 2003), and by pool-to-pool connectivity via

9

low-lying preferential flow paths (Price and Maloney, 1994; Quinton and Roulet, 1998). Such

fen complexes with gentle slopes and hydrological flow are better suited for water polishing

services (McCarter, 2016).

Both ombrotrophic bogs and ribbed fens are acidic, nutrient-deprived ecosystems

dominated by Sphagnum mosses (Riley, 2011; Sjörs, 1963). Their rates of productivity and

decomposition depend on the climate, degree of waterlogging, the nutrient composition and

nutrient availability within the peatland (Clymo and Hayward, 1982) as well as the dominant

Sphagnum species (Heal et al., 1978; Clymo, 1984). Both bogs and fens produce hummocks and

hollows, with intermediate carpets, each with its own assemblages of Sphagnum species (Andrus

et al., 1983). Hummock species, such as S. fuscum, have slower growth but are more recalcitrant

to decomposition as compared to hollow species (Clymo and Hayward, 1982; Johnson and

Damman, 1991; Turetsky et al., 2008). This recalcitrance to decay makes Sphagnum species the

predominant peat-formers in northern peatlands. Their peat forms the ridges in ribbed fens, so

their growth and decomposition in turn controls the hydrology of these peatland landscapes.

Sphagnum species are acid-generating (Clymo, 1963) and have a high cation-exchange

capacity relative to other plant life-forms, allowing them to efficiently scavenge for nutrients in

these nutrient-poor environments (Clymo and Hayward, 1982). However, nutrient enrichment

can change Sphagnum growth and decomposition and potentially hydrological relations in

peatlands, as has been found in numerous studies simulating enriched atmospheric N deposition.

Early European studies reported decreases in Sphagnum growth rates with enriched atmospheric

N deposition, and in some cases, Sphagnum mortality (Ferguson et al., 1984; Press et al., 1986;

Woodin and Lee, 1987; Verhoeven and Schmitz, 1991). Aerts et al., (1992) conducted a

fertilization experiment in European ombrotrophic bogs to simulate atmospheric N-enrichment

10

and found that the addition of N to peatlands with low atmospheric N deposition caused a

fourfold increase to the growth rate of Sphagnum. Those peatlands exposed to higher

atmospheric N deposition (>4 g m-2 year-1) were no longer N-limiting, but become P-limiting.

Other European peatland fertilization experiments have generally determined that high rates of

atmospheric N deposition cause a decrease in Sphagnum biomass and peat formation (Berendse

et al., 2001; Van Wijk et al., 2003). In North America, Bubier et al., (2007) conducted a five-

year nutrient addition experiment to an ombrotrophic bog in eastern Ontario using treatments

representative of elevated atmospheric N deposition (1.6 to 6.4 g m-2 yr-1), and some treatments

also added P (5 g m-2 yr-1) and K (6.3 g m-2 yr-1). Over the first two years, plant growth and net

ecosystem CO2 exchange (NEE) increased, indicating that the bog increased its carbon storage

capacity. By the third year and onward NEE levels began to decrease, as the plant community

shifted away from Sphagnum toward other mosses and vascular plants. After four years of

nutrient loading, the treatments with greater N loading, as well as P and K, had no more

Sphagnum cover because of competition for light with taller vascular plants such as shrubs

(Bubier et al., 2007). Larmola et al., (2013) concluded that the vegetation shifts, particularly the

loss of Sphagnum, is the key explanation why peatlands with longer-term nutrient enrichment

become weaker carbon sinks. However, most of these studies have focused on atmospheric

enrichment with low input rates, in ombrotrophic peatlands where the water is stagnant, and

often under warmer climates and greater anthropogenic influence as compared to the majority of

peatlands in boreal and subarctic landscapes. Limited research has been conducted in fens or

with higher enrichment rates or with point source inputs.

Kadlec (2009) conducted detailed studies of a wastewater polishing peatland running from

1970 to 2010 in Houghton Lake, Michigan. The municipality discharged point source additions

11

of wastewater into a 700 ha peatland seasonally (May-October) at a loading rate of 600,000 m3

year-1 with concentrations of 7.5 mg/L of dissolved inorganic nitrogen and 3.5 mg/L total

phosphorus (Kadlec, 2009). These enrichment levels far exceed that of the other peatland

fertilization studies focused on nutrient loadings simulating potential atmospheric deposition.

Within the first few years of wastewater discharge changes to vegetation composition were

significant, as Typha spp. began to increase in abundance and density to the point of invasion and

mass displacement of original peatland plant assemblages (Kadlec and Bevis, 2009). Peat

structure shifted from predominantly Sphagnum and sedge peat to floating mats of Typha spp.

with far faster rates of decomposition (Kadlec and Bevis, 2009).

Resource development pressures and the population growth of isolated communities have

increased in remote northern regions, such as in the HBL (Far North Science Advisory Panel,

2010). In consequence, peatland systems may be increasingly used to polish secondarily-treated

wastewater. There is a lack of understanding on the biological and hydrological response of

peatlands within northern subarctic climates, such as the HBL, to cope with high nutrient loading

at a point source resulting from anthropogenic developments.

In a collaborative study, McCarter (2016) provided insight on the hydrological response of

a subarctic ribbed fen receiving simulated secondarily-treated domestic wastewater from a

remote mining camp operation. This current study aims to provide insight on the biological

response to the point source nutrient loading by assessing the impact on the rates of productivity

and decomposition with key Sphagnum species, and the nutrient content and C:N ratios within

Sphagnum tissues. We hypothesized that the addition of simulated treated domestic wastewater

will, in the short-term, (i) increase the rate of productivity of peat-forming Sphagnum species;

(ii) increase the rate of decomposition of the peat; and (iii) increase nutrient content and decrease

12

C:N ratios of Sphagnum tissues. We also hypothesized that the production, nutrient uptake and

decomposition of hollow Sphagnum species would be more influenced by the nutrient additions

than hummock Sphagnum species, since they are at or near the water table.

Methods

We conducted the study near the De Beers Canada Victor Mine, within the Attawapiskat River

watershed of the Hudson Bay Lowland (HBL) in north-central Canada (52°49’08” N,

83°54’52” W; 80 m elevation). The HBL is a vast peatland plain underlain by glaciomarine

sediments and limestone bedrock (Martini, 2006). It is the world’s third largest peatland,

spanning 373 700 km2 (Abraham and Keddy, 2005), with average peat depths between 1-3 m

(Riley, 2011), making it a globally significant carbon sink with estimated storage of 20-30 g C

m-2 year-1 (Gorham et al., 2003). Hudson Bay and James Bay heavily influence this region,

creating a cool, humid, high-boreal climate, characterized by short cool summers and long cold

winters (Abraham and Keddy, 2005: Riley, 2011). The mean annual temperature is -1.3 °C, with

a mean of -22.3 °C in January, and 17.2 °C in July (Lansdowne House; 52°14’ N, 87° 53’ W;

280 Km WSW; 254 m elevation; 1971-2000 normals; Environment Canada, 2016). The mean

annual precipitation is 700 mm, with 291 mm falling during from June to the end of August. At

the Victor Mine, the 2015 growing season was wet, with 364 mm of precipitation from June 1 to

August 31, 2015, but only 157 mm during the same time period in 2016 (De Beers Canada,

unpublished data).

We studied two ribbed fens: an experimental fen (52°51’17” N, 83°56’35” W) and a

reference fen (52°47’00” N, 83°53’21” W; Figure 1). The fens are approximately 8.5 km apart

and drain into separate tributaries of the Attawapiskat River. The experimental fen has a mean

13

peat depth of 2.05 m, a total area of 9800 m2 (2240 m2 pools, 7560 m2 ridges), and an elevation

drop of 0.67 m across its 250 m length (McCarter, 2016). Its vegetation is split into two distinct

zones: a poor fen with distinct pool-ridge morphology (0-140 m), and a richer fen dominated by

carpets of Sphagnum rubellum and minimal ridge-pool morphology (140- 200 m; McCarter,

2016). The reference fen had similar peat depth and drop in elevation over its 150 m length, with

a distinct pool-ridge morphology throughout, and poor fen vegetation (McCarter, 2016).

McCarter (2016) added simulated domestic wastewater additions in 2014 and 2015.

Briefly, the experimental fen received a continual input of simulated wastewater from a point

source for 51 days in the summer 2014 and 41 days in summer 2015 at a rate of 38 m3 day-1. The

simulated wastewater was specially formulated to mimic the secondarily-treated wastewater

effluent from the Victor Mine camp or a small isolated community in northwestern Ontario

(McCarter, 2016), and contained SO4-2 (27.2 mg L-1), NO3

- (27.2 mg L-1), NH4+(9.1 mg L-1),

PO4-3 (7.4 mg L-1), K+ (24.5 mg L-1). A sodium chloride salt tracer was also added to the

experimental fen, so the simulated wastewater also contained Na+ (25.3 mg L-1) and Cl- (47.2 mg

L-1).

We established 72 sampling points in the experimental fen and 18 in the reference fen in

May 2015 using a stratified sampling design. Within the experimental fen, we first selected

sample zones up-gradient from the effluent discharge point (pool 0 and ridge 0) and down-

gradient, generally at each ridge and pool. Within each sampling zone, we selected three

sampling sites within a ridge in hummocks of Sphagnum fuscum, three along the edge of the

pools in carpets of Sphagnum rubellum, and three Sphagnum majus sampling sites within a pool.

We evenly distributed our sampling sites within each zone, and selected only undisturbed,

monospecific colonies. At each sampling site, we randomly chose three sample points. Within

14

the reference fen, we only sampled near the start and end of the fen, again with three sampling

sites in either zone in Sphagnum fuscum ridges, S. rubellum carpets along pools, and the aquatic

S. majus within the pools, with three randomly chosen points within each site.

To determine if there were temperature differences between microtopographical

positions, we placed HOBO® Pendant temperature loggers at random ridge and pool sample sites

throughout the experimental fen at a depth of 10 cm. Loggers recorded temperature every four

hours throughout the 2016 12-week growing season.

We measured the productivity of Sphagnum fuscum and S. rubellum using the crank wire

method (Clymo and Reddaway, 1974), with five replicate crank wires at each sampling point.

Briefly, we made the crank wires with 0.81 mm diameter (20 gauge) stainless steel wire bent into

a crank shape so that there was 10 cm of straight vertical wire on either side of a 1 cm long

horizontal cranked section. We inserted each crank wire into a Sphagnum carpet so that the

horizontal crank was level with the tops of the capitula and the remaining 10 cm of vertical wire

was suspended in the air. We measured productivity by measuring the length from the top of the

Sphagnum capitula to the top of the wire, using calipers. In 2015, we measured growth from July

23 to September 8 (6.5 weeks), and in 2016, from June 6 to September 2 (12 weeks). We

separated the 2016 season into two ~6-week periods, from June 6 to July 27 and from July 28 to

September 2, the second providing a comparable time fame to the 2015 productivity

measurements.

We determined the decomposition rate of S. fuscum and S. rubellum by means of mass loss

over time using the mesh decomposition bag technique (Johnson and Damman, 1991). Briefly,

we harvested Sphagnum strands from each sample point in the experimental fen and reference

fen in July 2015. We removed and discarded the top 1 cm of each strand including the capitulum,

15

and then cut each strand to 5 cm length and discarded the older, bottom portion of the strand to

ensure a constant age of Sphagnum strands. We determined their initial dry mass by placing ten

5 cm strands in the drying oven at 30 °C for 48 hours prior to weighing. We then placed the ten

strands in 0.2 mm diameter mesh nylon bags and heat-sealed them closed. We returned the

decomposition bags with the Sphagnum strands to the exact same sampling points in the field

from which they were harvested, with two replicate decomposition bags per sampling point, and

buried them at a depth of 10 cm below the surface. We also dried a subsample of each group of

Sphagnum strands at 70 °C for 48 hours to determine if there was any residual water and to

calculate a conversion factor between 30 °C to 70 °C drying temperatures. One year later in July

2016, we retrieved the decomposition bags from the field, rinsed them with deionized water to

remove external organic debris, and then oven dried them at 70 °C for 48 hours. We carefully

separated the remaining Sphagnum strands from the mesh bags and weighed them. We calculated

decomposition rates by correcting the initial mass at 30 °C using the calculated conversion

factor, then subtracting the final mass at 70 °C from initial mass at 70 °C. We divided the mass

loss decomposition data by each sampling location’s unique Sphagnum carpet density value,

which we previously determined by taking the mean number of Sphagnum strands per dm2 area

at each sampling zone.

To determine nutrient content within the Sphagnum, we collected samples of S. fuscum, S.

rubellum, and S. majus from the sampling locations in the Experimental and Reference Fens in

August of 2016. We removed the capitulum of each Sphagnum strand to have strictly non-living

(litter) components of the moss tested for nutrient analysis, and to remain consistent with the

procedures used in the growth and decomposition experiments. We air-dried samples for 30 days

at room temperature, ground them to a fine power using a ball mill grinder (RETSCH® Mixer

16

Mill 400), placed them into clean dry 20 ml glass scintillation vials, and sent them to the Ontario

Forestry Research Institute (OFRI) for nutrient analysis. They analyzed the samples for total

concentrations of C and N using an elemental combustion analyzer (Vario MAX Cube CN), total

S using a carbon/sulfur combustion analyzer (ELTRA® CS-800), and total P, K, Ca and Mg

using inductively coupled plasma (ICP; Genesis FEE ICP OES) run off a selenium dioxide

extraction following industrial method NO 786-86T (Bran and Luebbe, 1986).

We mapped the productivity and decomposition data of the experimental and reference

fens using size proportional bubble maps created in ArcGIS (ArcMap10.0, ESRI 2011). To

reduce the complexity of the nutrient data, we first conducted a principal component analyses of

the macronutrient variables within the Sphagnum plants (TN, TS, P, K, Ca and Mg), using

PRIMER® version 7. We used the first principal component (PC1) as well as the C:N ratio in

subsequent analyses. We then used a three-step approach to analyze the productivity,

decomposition and nutrient data. First, we conducted simple analyses of variance to determine

differences among species across all sample points. Second, we analyzed the growth,

decomposition, and nutrient data separately by species using a linear regression against distance

downgradient, with the fen as a categorical variable and their interaction. In the experimental

fen, we only included samples downgradient from the nutrient point source in the regression

analyses. We performed these analyses with Statistica® version 10, using a type I error rate of

5%, although we considered effects noteworthy with up to a 10% error rate. Third, we explored

for discontinuities in the shape of the individual regressions within the experimental fen to

identify breakpoints using segmented regression with the software SegReg®

(www.waterlog.info). Segmented regression models were only chosen if they were superior to

the linear regression models, as determined from their type I error rate. We also performed

17

Pearson correlation tests on the growth, decomposition, and nutrient data using SPSS®, (version

21, 2012).

Results

Surface temperatures within both ribbed fens increase over time throughout the duration of the

growing season (June to end of August) in both the 2015 and 2016 summers (Figure 2). In both

the experimental and reference fens the pools are consistently slightly warmer in temperature

than the ridges, by 2.5 to 3.5 oC over the growing season average, with median temperatures in

August, the hottest month, of 16.4 oC in pools and 14.2 oC in ridges.

The rates of productivity for both Sphagnum species were strongly correlated among the 6-

week and 12-week time periods in 2015 and 2016 (r > 0.80; Table 1). When we just consider the

productivity of both species across all sampling site distances in both fens, S. fuscum did not

differ in productivity from S. rubellum (2015 6-weeks: F1,41 = 0.62, P = 0.44; 2016 6-weeks:

F1,42 = 1.50, P = 0.23; 2016 12-weeks: F1,42 = 1.95, P = 0.17).

When we look at each species individually, the productivity of S. fuscum within the

experimental fen did not differ significantly from the reference fen over the 6-week periods in

the 2015 and 2016 growing seasons (both P = 0.79; Table 2; Figures 3 and 4) or over the 12-

week period in the 2016 growing season (P = 0.97; Table 2; Figures 5 and 6). The productivity

of S. fuscum also did not show a linear regression with distance downgradient in either fen over

any time period (Table 2), but when we examined for a segmented regression in the experimental

fen, the productivity of S. fuscum was slightly higher at all time periods near the point source and

dropped to until a common breakpoint of 48 m downgradient of the point source, after which

there was no change (2015 6-weeks: P = 0.065; 2016 6-weeks: P = 0.039; 2016 12-weeks:

P = 0.024; Figures 4 and 6).

18

In contrast, the productivity of S. rubellum within the experimental fen was significantly

greater than the reference fen at all time periods (2015 6-weeks: P = 0.014, 2016 6-weeks: P =

0.0.023, 2016 12-weeks: P = 0.019; Table 2; Figure 3 to 6). The productivity of S. rubellum did

not show a linear regression with distance downgradient over any time period, although a

borderline interaction occurred (Table 2), but when we examined for a segmented regression, the

productivity of S. rubellum was much greater at all time periods near the point source of nutrient

input until a common break point at 38 m from the point source (all P < 0.0001; Table 2), after

which, there was no change as productivity (Figures 4 and 6).

The one-year decomposition for both Sphagnum species did not significantly correlate with

any measures of productivity at any time (Table 1). Based on initial analysis of variance of the

data among species, the decomposition rates did not differ between S. fuscum and S. rubellum

(F1,37 = 2.04, P = 0.16). When we consider each species individually, the decomposition of S.

fuscum within the experimental fen did not differ significantly from the reference fen (P = 0.38;

Table 2; Figures 5 and 6). Its decomposition did not show a linear regression with distance

downgradient (P = 0.19; Table 2), with no interaction, and we found no segmented regression in

the experimental fen (P = 0.78). For S. rubellum, its decomposition was borderline higher in the

experimental fen than in the reference fen (P = 0.08; Table 2; Figures 5 and 6), but its

decomposition did not change with distance downgradient (P = 0.35), with no interaction (Table

2), and we found no segmented regression in the experimental fen (P = 0.12).

The first principal component (PC1) of nutrient content within the Sphagnum tissues

explained 74% of the total variation, while the second component (PC2) only explained 17% of

the variation (Figure 7). PC1 was strongly positively correlated with TN, TS, TP, K, and Mg,

while PC2 was strongly but negatively correlated with Ca (Table 2). This PC1 variable was

19

moderately to strongly correlated to C:N ratio within each species (S. fuscum: r = 0.53; S.

rubellum: r = 0.81; S. majus: r = 0.83).

All three Sphagnum species had significantly different nutrient contents in their tissues as

determined by their PC1 values (F2,65 = 37.75, P < 0.0001, Figure 8A), with S. fuscum and S.

rubellum differing from each other slightly (P = 0.020) and S. majus differing strongly from both

these species (P = 0.0001). For S. fuscum, PC1 of plant nutrient content did not differ

significantly between fens, there was no effect of distance, and no interaction (Table 3). S.

fuscum also showed no segmented regressions of PC1 with distance within the experimental fen.

Likewise, the C:N ratio of S. fuscum did not differ between fens, again with no regression against

distance, no interaction (Table 3; Figure 8B) and no segmented regression.

For S. rubellum, PC1 of plant nutrient content was borderline higher in the experimental

fen (P = 0.072), indicating more plant nutrient content in the experimental fen, but there was no

effect of distance and no fen by distance interaction (Table 3; Figure 8A). S. rubellum also

showed no segmented regression. C:N ratio of S. rubellum did not differ between fens, again had

no regression with distance, no fen by distance interaction (Table 3), and no segmented

regression (Figure 8B).

The PC1 of plant nutrient content for S. majus was much higher in the experimental fen

than the reference fens (P = 0.0002; Table 3; Figure 8A), and there was a significant negative

regression with distance downgradient (P = 0.045). A borderline significant fen by distance

interaction (P = 0.07) shows that the negative regression with distance only occurred in the

experimental fen (experimental: P = 0.004; reference: P = 0.55). PC1 showed no segmented

regression, thus a simple linear regression fits the data best. The C:N ratio of S. majus was

significantly lower in the experimental than in the reference fen (P = 0.0002; Table 3; Figure

20

8B). C:N ratio increased significantly with distance downgradient in both fens (P = 0.045), with

no interaction, suggesting that both the experimental and reference fen show an increasing C:N

trend with increasing distance downgradient (Table 3; Figure 8), however, when C:N ratios were

analyzed separately for each fen, only the experimental fen showed significant regression with

distance (experimental: P < 0.0001; reference P = 0.55). C:N ratio for S. majus also showed no

segmented regression (Figure 8).

Within the experimental fen, S. fuscum PC1 and C:N ratios significantly correlated with

the 2015 six-week productivity rates (PC1: r = 0.559, P = 0.038; C:N: r = -0.69, P = 0.006), but,

did not significantly correlate with productivity during the 2016 season (Table 1). For S.

rubellum in the experimental fen, only PC1 significantly correlated with 2015 6-week

productivity rates (r = 0.741, P = 0.004; Table 1). PC1 and C:N ratios for S. fuscum or S.

rubellum did not correlate with decomposition at any time (Table 1).

Discussion

We examined changes in Sphagnum productivity and decomposition rates following point

source press applications of treated domestic wastewater, as well as compare uptake of nutrients

across the hydrological flow gradient of the experimental fen.

We had first hypothesized that the point source addition of simulated treated wastewater

would increase Sphagnum productivity within the experimental fen, and that the carpet species

would show a greater productivity response to the nutrient additions than the hummock species.

Our experimental results supported our hypothesis. The lower-lying S. rubellum showed

significantly greater productivity rates within the experimental fen compared to reference fen

levels, and S. fuscum, the higher hummock species, showed little to no growth response with

21

added nutrients and maintained similar productivity levels throughout both fens. These results

support other research conducted on Sphagnum productivity rates in northern low-nutrient

peatlands (Turetsky et al., 2008), and northern peatlands exposed to increased nutrient

fertilization (Aerts et al., 1992; Aerts et al., 2001).

Soil moisture is a key environmental factor that influences Sphagnum productivity (Clymo

and Hayward, 1982). The difference in growth response between the two species may be due to

their difference in topographic positions, and water saturation level. The higher elevation of the

peat ridges resulted in S. fuscum having less access to the nutrient- enriched water. Hummock

species, such as S. fuscum are well adapted to dry conditions (Andrus, 1986). In period of

drought and lower water table level, S. fuscum will use passive transport to conduct water though

capillary uptake (Clymo and Hayward, 1982; Thompson and Waddington, 2008 Precipitation

levels were high and relatively constant throughout the 2015 growing season (McCarter, 2016),

therefore, S. fuscum may have retained water through retention of precipitation water rather than

from increased pore-water pressure causing passive capillary uptake of the fertilized water.

Therefore, the greater precipitation levels and the ridge’s height above the fertilized water table,

help explain why there was no difference in S. fuscum productivity rates between the

experimental and reference fens in 2015 or 2016 (Table 2), and why the nutrient content (PC1

and C:N ratio) of surface level S. fuscum was also not significantly different between fens .

S. rubellum, in contrast, grows in dense carpets closer to the water table (Clymo and

Hayward, 1982). Our results showed S. rubellum had significantly greater productivity within the

first 50 m than the reference fen levels in 2015 and in 2016. Water table levels, and surface water

discharge in the experimental fen were average throughout 2014, however, were very high

throughout summer of 2015 (McCarter, 2016). In 2015, the first few ponds and ridges of the

22

experimental fen were experiencing high hydrological connectivity (McCarter, 2017). The pore

water saturating the low-lying S. rubellum within the experimental fen would have contained

more of the added nutrients, explaining the greater growth of S. rubellum strands within the first

50 m away from the point source of nutrient input. We suspect that the low-lying preferential

flow paths connecting Pool 1, and Pool 2 allowed for transportation and distribution of the

elevated nutrient water to reach all S. rubellum sample sites within the first 50 m of the

experimental fen. S. rubellum, being a carpet-forming species, has high shoot and spreading

branch density (Clymo and Hayward, 1982), and thus excellent water retention capabilities

(Rydin and McDonald, 1985). High water retention capability would allow for continued uptake

of nutrients into the new-grown tissues accounting for the continued high growth rates

throughout the 2016 growing season and higher nutrient content.

We had also hypothesized that increasing nutrients within the experimental fen would

increase the rate of Sphagnum decomposition, however, we did not find a significant difference

in decay rates between the two sites. Our decomposition results did not support our hypothesis.

Many environmental factors influence Sphagnum decomposition rates, for example,

temperature (Sjörs, 1959), microbial community (Thormann et al., 2004), degree of oxygenation

(Johnson and Damman, 1993), litter chemistry, and nutrient availability (Aerts et al., 1992).

These environmental factors also differ between hummock, carpet, and hollow habitats (Andrus

et al., 1983). For example, hummock and hollow Sphagnum species differ in chemical

composition where hummock species contain more complex recalcitrant organic matter (uronic

acids and polyphenolic compounds) than hollow species (Clymo, 1963; Kälviäinen and Karunen,

1984; Johnson and Damman, 1991). Therefore, this variation in litter chemistry as well as the

variation in moisture gradient between hummock and flat carpet Sphagnum habitats led us to

23

hypothesize that S. rubellum would have greater mass loss after the year incubation than S.

fuscum in both fens. As nitrogen content within Sphagnum litter increases, the decay rate also

increases (Clymo, 1965; Heal et al., 1978; Coulson and Butterfield, 1978). Therefore, we

predicted that because the experimental fen was also receiving high nutrient loading, the

increased N would only exaggerate this increase in mass loss to decomposition of S. rubellum

litter.

We found that within both fens S. fuscum and S. rubellum decomposed at similar rates.

Mean S. rubellum decomposition rate within the experimental fen (1.6 g dm-2 year-1) was slightly

greater than the reference fen mean (1 g dm-2 year-1). Coulson and Butterfield (1978), found that

increasing the nitrogen content within the living Sphagnum plants, increases the rate of decay

when the plants die. But ultimately, Johnson and Damman (1993) found that the litter chemical

composition is what limits decay regardless of the microhabitat (moisture or oxygenation level)

that the litter is subjected to. Perhaps the S. fuscum and S. rubellum litter chemistry and nutrient

content was not different enough from one and other to make a significant difference in the

decay rates because the Sphagnum tissues collected for the decomposition received only one year

of nutrient exposure prior to harvest, while the Sphagnum tissues harvested for nutrient analysis

were exposed to two years of nutrient additions.

The pattern of nutrient content within the 2016 Sphagnum tissues support our nutrient

content hypothesis. S. fuscum nutrient levels showed no difference between fens, S. rubellum

showed border line significant difference between fens, and S. majus showed a strong significant

increase in nutrient content within the experimental fen. This pattern indicates that nutrient

uptake and retention was strongly linked to the ecological moisture gradient from hummock to

hollow (less saturated to fully saturated). This result was expected because the nutrients were

24

added directly to the surface water table level, therefore, the Sphagnum species with the closest

interaction with the water table would have the greatest ability to uptake those nutrients. This

result may not have occurred if the nutrient additions were deposited aerially and evenly

throughout the fen, as the case would be with peatland fertilization experiments that simulate

atmospheric deposition of nutrients.

When assessing productivity rates across the hydrological gradient (distance from the point

source), we can see that the zone most impacted by the added nutrients ends at about the 50 m

mark. This distance covers from Pool 1 to the third ridge, which indicates that sample locations

beyond the third ridge may not be receiving elevated levels of nutrients. Our nutrient data

confirms that beyond the 70 - 100 m mark within the experimental fen, nutrient concentrations

remain comparable to reference fen levels. These results parallel that of the hydrological

transport of the wastewater nutrients within the pore water found by McCarter (2016). Our

observed plant ecological response to the added wastewater nutrient can confirm that the pool-

ridge-pool morphology of these ribbed fens appears to successfully polish and immobilize the

added nutrients.

Throughout this experiment, Sphagnum productivity remained greater than the rate of

Sphagnum decay. This result was exaggerated within the first 50 m downstream from the

nutrient input, as the rate of productivity far exceeded the rate of decay. Therefore, an increase in

nutrients leads to an increase in formation of peat, which translates to an increase in carbon

storage. Increasing the capacity for these northern peatlands to store greenhouse gases would

enhance an existing valuable ecosystem service. However, our results are an indication of the

short-term ecological trends within these subarctic peatlands. Other peatland fertilization

experiments, which maintained long-term nutrient additions have concluded that Sphagnum

25

decay rates increase with increasing nutrient content (Clymo, 1965; Heal et al., 1978), and in the

long-term (> 5 years), Sphagnum decline in abundance, as they become out competed by other

vascular plants, such as grasses and shrubs (Bubier et al., 2007; Bragazza et al., 2004; Berendse

et al., 2001).

The key question remaining is how long can the northern peatlands of the HBL

successfully uptake the added nutrients and remain Sphagnum dominant? Further research into

the length of time these peatlands can sustain high nutrient loading before significant changes to

the vegetation community would be highly beneficial. Additional monitoring of the productivity

decomposition of the main peat forming vegetation would be useful, as would direct monitoring

of the carbon flux within these peatlands if they are to be exposed to high nutrient loading long

term. It is important for the HBL landscape to remain a globally significant carbon storage

reservoir rather than switch to becoming a carbon source. The biological evidence provided by

this study, as well as the hydrological evidence provided by McCarter (2016), suggests

feasibility of these northern peatlands to polish treated domestic wastewater. Collaborators are

conducting further research on the biogeochemical interactions resulting from adding wastewater

into these peatlands, and will provide additional evidence towards assessing the environmental

suitability of using the HBL peatlands to polish domestic wastewater.

Conclusion

Our findings suggest that Sphagnum within the HBL peatlands were nutrient deprived as

providing them with additional nutrients over the course of two growing seasons allowed for

increased productivity with no significant rise in decomposition. Variations in soil moisture level

and microtopographic position relative to the water table plays a large role in accounting for the

26

differences in growth response and nutrient uptake between the Sphagnum species. Low-lying

Sphagnum species, such as the dominant carpet and hollow species, were responsible for the

uptake and immobilization of the nutrients.

Short-term high nutrient loading of the northern ribbed fen increased the formation of

highly recalcitrant peat, indicating the potential for enhancement of the carbon sequestration

ecosystem service, which the HBL peatlands are well known for providing. However, the long-

term ecological balance between primary productivity and decomposition for this landscape

remains speculative. Further research on the time line for maintaining a recalcitrant Sphagnum

dominant peatland following press nutrient applications is required, along with investigation of

possible biogeochemical interactions associated with the addition of nutrients.

Acknowledgements

The research was funded through the NSERC Canadian Network for Aquatic Ecosystem

Services (CNAES) and De Beers Canada. The first author gratefully acknowledges the salary

support through the NSERC Industrial Postgraduate Scholarship program. We are also grateful

for travel support provided by the Northern Scientific Training Program.

We thank field assistants Angela Borynec and Ainsley Davison, graduate students Andrea

Hanson and Brittany Rantala-Sykes and network collaborators Drs. Colin McCarter, Jonathan

Price, and Brian Branfireun. We sincerely thank all employees at the De Beers Victor Mine who

assisted us along the way, especially Brian Steinback, Stephen Monninger, Terry Ternes, Anne

Boucher, Rod Blake, Nick Gagnon, Jake Carter and all members of the Environment and

Reclamation department. We are also very grateful for the laboratory analysis and analytical

services provided by Scott Bowman and Maara Packalen from the Ontario Forestry Research

27

Institute. We would like to acknowledge the help of committee members Dr. N. Basiliko and Dr.

G. Spiers for constructive criticism.

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Thormann, M. N., Bayley, S. E., & Currah, R. S. (2004). Microcosm tests of the effects of

temperature and microbial species number on the decomposition of Carex aquatilis and Sphagnum fuscum litter from southern boreal peatlands. Canadian Journal of Microbiology, 50(10), 793-802.

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resource allocation among moss species control decomposition in boreal peatlands. Journal of Ecology, 96(6), 1297-1305.

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31

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dynamics since the Last Glacial Maximum. Geophys. Res. Lett., 37, L13402, doi:10.1029/2010GL043584.

32

Table 1. Spearman’s correlation coefficients among Sphagnum productivity, decomposition, first principal component (PC1) of the

nutrient data and the C:N ratio within Sphagnum collected from the experimental fen. S. fuscum is shown in the upper triangular

matrix (grey) and S. rubellum in the lower triangular matrix (white). Correlation coefficients in bold are significant at P < 0.05 based

on a two-tailed test.

Productivity 2015

6 weeks

Productivity 2016

6 weeks

Productivity 2016

12 weeks

Decomposition

1 year

PC1 C:N

ratio

Productivity 2015: 6 weeks - 0.652 0.600 0.018 0.559 -0.692

Productivity 2016: 6 weeks 0.803 - 0.914 0.304 0.094 -0.332

Productivity 2016: 12 weeks 0.838 0.982 - 0.305 0.096 -0.308

Decomposition: 1 year 0.369 0.072 0.140 - -0.138 -0.076

PC1 0.741 0.359 0.289 0.034 - -0.791

C:N ratio -0.463 -0.253 -0.214 -0.056 -0.853 -

33

Table 2. Analyses of variance for productivity and decomposition separated by species, as a function of the fen site (experimental or

reference), distance downgradient and their interaction. Results with type I error < 10% are in bold.

Productivity

2015 (6-weeks)

Productivity

2016 (6-weeks)

Productivity

2016 (12-weeks)

Decomposition

2015-2016

Species Source df MS F P

MS F P

MS F P

MS F P S. fuscum Fen 1 0.02 0.1 0.787 0.17 0.1 0.791 0.01 0.0 0.973 0.16 0.8 0.387

Distance 1 0.49 2.1 0.169

2.72 1.2 0.289

2.75 0.6 0.452

0.39 1.9 0.190

Fen*Distance 1 0.10 0.4 0.530

0.13 0.1 0.812

1.18 0.3 0.620

0.01 0.0 0.829

Error 16 0.24 2.27 4.63 0.21 S. rubellum Fen 1 18.92 7.9 0.014

41.41 6.4 0.023

71.22 7.0 0.019

1.35 3.5 0.082

Distance 1 2.85 1.2 0.294

16.98 2.6 0.127

26.31 2.6 0.129

0.36 0.9 0.353

Fen*Distance 1 8.57 3.6 0.079

14.60 2.2 0.155

27.36 2.7 0.122

0.55 1.4 0.254

Error 15 2.39 6.50 10.21 0.39

34

Table 3. Correlation (loadings) between the original variables and the first two principal

components from the principal component analysis (PCA). Correlations in bold are significant at

a 5% type I error rate.

PC 1 PC 2

TN 0.953 -0.042

TS 0.949 0.034

P 0.961 0.107

K 0.957 0.005

Ca -0.020 -0.998

Mg 0.898 -0.133

35

Table 4. Analyses of variance for the first principal component of plant nutrient content and for

the C:N ratio, separated by species, as a function of fen site (experimental or reference), distance

downgradient, and their interaction. Results with type I error < 10% are bolded.

PC1 C : N Species Source df MS F P

MS F P

S. fuscum Distance 1 0.018 0.20 0.659

69.5 0.17 0.681

Fen 1 0.010 0.11 0.741

74.4 0.19 0.670

Fen*Distance 1 0.134 1.53 0.232

483.6 1.22 0.285

Error 18 0.087

397.5 S. rubellum Distance 1 0.29 0.45 0.513

1145.4 2.17 0.160

Fen 1 2.42 3.70 0.072

621.5 1.18 0.294

Fen*Distance 1 1.29 1.96 0.180

186.7 0.35 0.560

Error 16 0.65

528.2 S. majus Distance 1 10.7 4.74 0.045

664.3 7.76 0.013

Fen 1 49.7 21.99 0.0002

2005.6 23.44 0.0002

Fen*Distance 1 8.0 3.56 0.077

89.5 1.05 0.322

Error 16 2.3 85.5

36

Figure 1. Google Earth satellite image of the De Beers Victor Mine site (June 2013),

showing the experimental fen and the reference fen. The studied fens are 8.5 km apart.

6.0 km

Attawapiskat River

Experimental Fen

Reference Fen

N

37

Figure 2. Box plots of monthly temperature of Pond 1(white) and Ridge 1 (grey) at 10 cm depth

within the experimental fen during the 2016 growing season.

Month

AugustJulyJune

30

25

20

15

10

5

0 Pool Ridge Pool Ridge Pool Ridge

Tem

pera

ture

Co

38

Figure 3. The productivity of Sphagnum fuscum (left) and Sphagnum rubellum (right) over a six-week period in the summer of 2015

(top) and the summer of 2016 (bottom). Direction of hydrological flow runs from top to bottom within each fen, following the

sequence of pool numbers. The arrow on Pool 1 of the experimental fen marks the location of the point source of nutrient addition.

The reference fen is 8.5 km away from the experimental fen, but they are shown together here.

39

Figure 4. Productivity over 6 weeks during the growing season in 2015 (top) versus 2016

(bottom) for Sphagnum fuscum (left) and Sphagnum rubellum (right) as a function of distance

from the discharge point in the experimental fen and the top edge of the fen in the reference fen.

Solid lines and solid circles represent experimental fen data, and open circles and dashed lines

represent the reference fen data. The vertical dotted line marks the point source input of nutrients

in the experimental fen. Samples with negative distance values are located upgradient from the

nutrient discharge point.

Pro

duct

ivity

(g d

m

6 w

eeks

)

Distance from point source (m)

0

2

4

6

8

10 Sphagnum fuscum2015

0

2

4

6

8

10

-50 0 50 100 150 200 250

Sphagnum rubellum2015

-50 0 50 100 150 200

Sphagnum fuscum2016

Sphagnum rubellum2016

250

-2-1

40

Figure 5. The productivity of Sphagnum fuscum (top left) and Sphagnum rubellum (top right) over a 12-week period in the

summer of 2016, and the decomposition rate of S. fuscum (bottom left) and S. rubellum (bottom right) over one year from July

2015 to July 2016. Direction of hydrological flow runs from top to bottom within each fen, following the sequence of pool

numbers. The arrow on Pool 1 of the experimental fen marks the discharge point source for nutrient addition. The reference fen is

8.5 km away from the experimental fen, but they are shown together here.

41

Figure 6. Linear regression plots of the productivity over 12 weeks during the growing season in

2016 (top) and annual decomposition (bottom) for Sphagnum fuscum (left) and Sphagnum

rubellum (right) in the experimental fen (solid circles and lines) and the reference fen (open

circles and dashed lines). The vertical dotted line marks the location of the discharge point of

nutrients, and negative distance values correspond to sample sites up gradient from the point

source.


0

2

4

6

8

-50 0 50 100 150 200 250

Dec

ompo

sitio

n(g

dm

ye

ar )

-50 0 50 100 150 200 250

0

2

4

6

8

10

12

14

Pro

duct

ivity

(g d

m

12 w

eeks

)

Sphagnum fuscum Sphagnum rubellum

Sphagnum fuscum Sphagnum rubellum

-2-2

-1-1

42

Figure 7. Principal component ordination plot of the nutrient content of all three Sphagnum

species in the experimental fen (solid) and reference fen (open). The percent variation explained

by principal component is shown in parentheses on each axis. Together they summarize 91% of

the total variation of the nutrient content in the Sphagnum species.

-2 0 2 4 6 8

PC 1 (74%)

-4

-2

0

2P

C 2

(17%

)

S. fuscumS. rubellumS. majus

Exp Ref

43

Figure 8. Linear regression plots of (A) the first principal component of Sphagnum nutrient

content (PC1; left) and (B) the C:N ratio (right) in the experimental fen (solid circles and lines)

and the reference fen (open circles and dashed lines). The vertical dotted line marks the location

of the discharge point of nutrients, and negative distance values correspond to sample sites up

gradient from the point source.

-4

-2

0

2

4

6

8

-4

-2

0

2

4

6

8

PC1

-4

-2

0

2

4

6

8

-50 0 50 100 150 200 250


S. fuscum

S. rubellum

S. majusC

: N

0

50

100

150

0

50

100

150

-50 0 50 100 150 200 250


0

50

100

150

S. majus

S. rubellum

S. fuscum

A B

44

CHAPTER 2: Decomposition of Sphagnum peat from a ribbed fen receiving simulated treated wastewater: an incubation experiment along a nutrient loading gradient

Amanda Lavallee1,2 and Daniel Campbell1,3

1 Vale Living with Lakes Centre, Laurentian University, Sudbury, Ontario, Canada P3E 2C6

2 Department of Biology, Laurentian University, Sudbury, Ontario, Canada P3E 2C6

3 School of the Environment Laurentian University, Sudbury, Ontario, Canada P3E 2C6

45

Abstract

Northern peatlands are dominated by Sphagnum moss. Slow decomposition in these peatlands

leads to the sequestration and burial of carbon, providing a globally key ecosystem function.

Resource extraction industries and small communities are growing in some northern peatland-

dominated regions, so there is increasing interest in harnessing peatlands to polish wastewaters.

In a previous collaborative research project, we set up a large-scale field experiment in the

subarctic Hudson Bay Lowland to evaluate the effects of point-source additions of simulated

treated wastewater into a ribbed fen, and we found a large productivity effect but no apparent

impact on decomposition. We used a factorial peat incubation experiment to further investigate

the decomposability of three dominant Sphagnum species representing hummock, carpet and

hollow microsites. We tested whether these Sphagnum peats originating from the start versus the

end of the experimental fen decomposed at different rates than in a reference fen. We also tested

whether the nutrient dose, ranging from distilled water to up to ten times the dose in the field

experiment, affected decomposition. We incubated samples under anaerobic conditions for 40

days, then aerobic conditions for 24 hours, then measured decomposability via microbial CO2

and CH4 production. Results show that hollow Sphagnum species consistently released more

greenhouse gases than carpet or hummock species. Peat originating from the nutrient-enriched

start of the experimental fen also had significantly greater emissions of CO2 and CH4. However,

the addition of nutrients to the incubation jars did not significantly increase CO2 or CH4

production, and thus did not increase decay potentials. This suggests that Sphagnum peat from

northern regions may be tolerant to high nutrient loadings associated with treated domestic

wastewater and can retain their ability to resist decomposition, leading to continued carbon

storage and net sequestration of greenhouse gasses.

46

Key words: Microbial respiration, methane production, carbon dioxide production, wastewater

polishing, string fen, peatland, Hudson Bay Lowland, peatland microtopography

47

Introduction

Northern peatlands sequester carbon because their rate of primary productivity exceeds their rate

of decomposition (Clymo and Hayward, 1982) and Sphagnum mosses are the key peat-forming

species. Their slow decomposition leads to the sequestration and burial of carbon (Roulet, 2000)

and provides a globally key ecosystem function. Sphagnum mosses occur across

microtopographical gradients, from raised hummocks to flat carpets or lawns to depressed

hollows or pools, in different species assemblages and under different decomposition

environments (Andrus et al., 1983; Clymo and Hayward, 1982).

The decomposition of Sphagnum peat is first influenced by temperature (Sjörs, 1959;

Thormann et al., 2004). Northern environments have discontinuous or continuous permafrost

(Riley, 2011), leading to inherently slow rates of decay in northern peatlands (Bartsch and

Moore, 1985; Johnson and Damman, 1993; Puranen et al., 1999). Warmer soil temperatures

increase the rates of peat decay (Davidson et al., 2000; Ise et al., 2008). An incubation study

using northern peat found that an increase in temperature from 12 °C to 22 °C caused up to a

fourfold increase in CH4, and a twofold increase in CO2 production (Yavitt et al., 1997). Peat

warming and decomposition remains a main concern for climate change, although little

consensus exists on the directional impacts on peat accumulation with climate warming

(Davidson and Janssens, 2006).

Second, Sphagnum peat decomposition is heavily influenced by the water level and the

consequent degree of aeration and redox potential (Yavitt et al., 1997). Peat decomposes two to

three times faster under aerobic conditions compared to anaerobic conditions (Moore and Dalva,

1997; Bridgham and Richardson, 1992). The uppermost layer within a peatland is the acrotelm,

which has fluctuating water table levels with at least periodic aerobic conditions, while the

48

underlying layer is the catotelm, which is fully saturated, with anaerobic conditions (Ivanov,

1981). Hummocks have a thick acrotelm (20-50 cm), while carpets are intermediate (5-20 cm)

with intermittent water saturation, and hollows have little or no acrotolem (0-5 cm; Rydin and

Jeglum, 2013). Peat is largely decomposed by aerobic or facultatively anaerobic bacteria and

fungi, which metabolize the Sphagnum litter (Clymo and Hayward, 1982; Thormann et al.,

2004), producing CO2 or CH4 as end products. CH4 production (methanogenesis) occurs almost

exclusively under low aerobic or anaerobic conditions, often just below the water table within

the peat profile (Nillson and Bohlin, 1993; Bubier and Moore, 1994). Measuring the amount of

microbial CO2 and CH4 production from peat has been widely used to quantify the rate of peat

decomposition (Singh and Gupta, 1977).

Third, the chemistry of the Sphagnum litter plays a large role in its decomposition (Andrus,

1986; Bragazza et al., 2006). Hummock species contain a greater concentration of complex

highly-recalcitrant uronic acids and polyphenolic compounds than hollow species (Clymo, 1963;

Kälviäinen and Karunen, 1984; Johnson and Damman, 1991; Turetsky et al., 2008), so hummock

species decompose more slowly (Clymo and Hayward, 1982; Andrus, 1986; Johnson and

Damman, 1991; Hogg, 1993). Furthermore, younger Sphagnum litter near the surface decays

more rapidly than deeper and older litter because the younger material has more easily

degradable organic compounds, leaving the more complex and highly recalcitrant compounds in

deeper peat layers (Clymo, 1984; Johnson and Damman, 1991; Hogg et al., 1992; Hogg, 1993).

The degree of nutrient uptake within the new growth and fresher Sphagnum tissues has a strong

impact on the decay potential of peat material as it becomes litter (Bartsch and Moore, 1985;

Johnson and Damman, 1993; Hogg, 1993).

49

Finally, nutrients are in short supply in northern peatlands (Riley, 2011), so their addition,

particularly of nitrogen, have been shown to intensify and increase the rates of Sphagnum

decomposition (Berendse et al. 2001; Van Wijk et al. 2004). Laboratory incubations of peat

have found that N and P additions increase the decay potential of Sphagnum peat (Hogg et al.

1994). In a four-year field experiment, however, Aerts et al., (2001) found that N or P additions

had little effect on CO2 production at high or low N sites. In another experiment, after five years

of peatland fertilization, Sphagnum biomass and peat formation were significantly reduced

(Bubier et al. 2007; Larmola et al. 2013). Ambiguities remain regarding the effects of nutrient

additions on the rate of Sphagnum decay, and results differ depending on the concentration of

nutrient amendment, temperature, oxygenation and water saturation level, differences in litter

chemistry, and particularly, variations between Sphagnum species (Aerts et al., 1992; Bridgham

and Richardson, 1992; Yavitt et al., 1997; Turetsky et al., 2008).

Northern peatlands provide other regulating ecosystem services of direct benefit to humans

besides carbon sequestration and burial, including climate regulation, erosion protection, water

quantity regulation and water filtration and purification (Kimmel and Mander 2010). We have

commonly used the filtering function of marshes to polish treated domestic wastewaters (Kadlec

and Wallace 2009), but we have less often used northern peatlands to polish wastewater (Kadlec

2009; Ronkanen and Klove 2009). Resource extraction industries and small communities are

growing in some peatland-dominated regions (Far North Science Advisory Panel 2010), so there

is increasing interest in harnessing peatlands to polish wastewaters.

Along with collaborative partners, we set up a large-scale field experiment in the subarctic

Hudson Bay Lowland to evaluate the effects point source additions of simulated treated

wastewater into a ribbed fen (McCarter, 2016). The fen effectively polished the nutrients over a

50

two-year period (McCarter, 2016). Sphagnum growth was strong in response, but we found no

apparent impact of the increased nutrient loading on decomposition in the field (Chapter 1 in this

thesis). Would the peat produced in this experimental fen decompose more or less rapidly if we

changed the nutrient dose? If decomposition rates increased with higher nutrient amendments,

this could have large consequences on the hydrology, structure, composition and water polishing

function of treatment fens.

We used a laboratory incubation study to determine the influence of peat origin in the

experimental fen versus subsequent nutrient loading on the decay potentials for three species of

Sphagnum representing hummock, carpet and hollow environments. We hypothesized that a

hummock-forming species (Sphagnum fuscum) would decay more slowly than a carpet species

(Sphagnum rubellum), followed by a hollow species (Sphagnum majus). We then hypothesized

that, following two seasons of high nutrient loading, peat originating from the start of the

experimental fen would decay most rapidly, producing greater amounts of CO2 and CH4 than

peat originating from the end of the experimental fen and a reference fen. Finally, we

hypothesized that increasing nutrient amendments would further stimulate Sphagnum

decomposition and CO2 and CH4 emissions, up to a certain threshold.

Methods

We collected the peat for this incubation experiment from an experimental ribbed fen receiving

simulated secondarily-treated wastewater, near the De Beers Canada Victor Mine, located within

the Attawapiskat River watershed of the Hudson Bay Lowland (HBL) in north-central subarctic

Canada (52°49’08” N, 83°54’52” W; 80 m elevation). Details of the site and the experimental

fen can be found in McCarter and Price (2017), and in Chapter 1 of this thesis. The simulated

51

wastewater was specially formulated to mimic the secondarily-treated wastewater effluent from

the Victor Mine camp, and would also be typical of a small isolated community in northwestern

Ontario. Briefly, the experimental fen received a continual input of simulated wastewater from a

point source for 51 days in the summer 2014 and 41 days in summer 2015 at a rate of

38 m3 day-1. The field loading concentrations of nutrients were 27.2 mg L-1 of NO3-, 9.1 mg L-1

of NH4+, 7.4 mg L-1 of PO4

-3, 24.5 mg L-1 of K+ and 27.2 mg L-1 of SO4-2. A sodium chloride salt

tracer was also added, so the experimental fen also received 25.3 mg L-1 of Na+ and 47.2

mg L-1of Cl- (McCarter, 2016).

We collected surface peat (<~12 cm) on August 29 and 30, 2015 within three origin zones:

the start of the experimental fen (Start EXP), the end of the experimental fen (End EXP), and

from a nearby reference fen (REF). Surface water concentrations of NO3-, NH4

+, PO4-3, and

SO4-2 remained elevated up to 50 m from the simulated wastewater point source (McCarter

2016), so we collected the Start EXP peat within the first 50 m downgradient from the point

source, in and around the first two pool-ridge sequences. We collected End EXP peat from zones

140 to 250 m from the point source, because it had low nutrient concentration in surface waters,

comparable to the reference fen. We collected REF peat samples close to the end of the reference

fen. Within each zone, we collected peat from Sphagnum fuscum hummocks, S. rubellum carpets

and S. majus pool habitats. We collected six samples per species per origin zone and bulked them

together in plastic bags. We transported them cold to Laurentian University and kept them

refrigerated for 5 weeks until we began the experiment. Prior to incubation experiment, we

sorted through the material and removed and discarded any living Sphagnum sections of the

strands to ensure that we only used dead (non-photosynthesizing) Sphagnum peat.

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We set up a factorial experiment with the three species (S. fuscum, S. rubellum, and S.

majus), three peat origins (Start EXP, End EXP, and REF) and five levels of nutrient

amendments (0x, 0.5x, 1x, 2x, and 10x) of the loading levels in the field experiment. We used

distilled water for the 0x control treatment and we diluted a concentrate of the same nutrient mix

used in the field to obtain the other nutrient dosages. We conducted the incubations in 250 mL

mason jars with tight sealing lids containing a rubber stopper in the centre to allow for gas

extraction with a needle and syringe. We placed 10 g of fresh peat material into each jar, with 40

mL of the corresponding nutrient solution.

We first conducted a 40-day anaerobic incubation to simulate flooded conditions. We

sealed the jars tightly to make the jar environment anoxic. We determined the remaining

headspace volume of each individual jar, and then we flushed the headspace of each jar with N2

gas using a Yellow JacketÒ vacuum pump (SuperEvac 8) to ensure there was no other gases

present prior to incubation. We placed the jars in a BioChambers® growth chamber (model

AC-60), with no light and set to a constant 15 °C, which was the average air temperature during

the 2015 growing season at De Beers Victor Mine. We extracted samples on days 1, 3, 7, 17 and

40, although we only present the day 40 results here. To extract the gas, we used 10 mL plastic

syringes with needles to pierce through the rubber stopper in the jar lids. We first injected 10 ml

of N2 gas into a jar, flushed the syringe three times and then drew 10 ml of gas from the sample

into the syringe. We ran the gas samples through a SRI model 8610C Gas ChromatographÒ using

PeakSimpleÒ software within 48 hours after each sampling event. We also ran a standard with

known CO2 and CH4 every 10 samples for quality control measures. Using the gas standards as a

conversion factor and the known headspace volume within each jar, we used the ideal gas law

equation to solve for the mass of CO2 and CH4 gas produced within each jar. We corrected our

53

data to account for the dilution factor generated by adding 10 mL of N2 gas to the headspace of

each jar at each sampling event.

On day 41, we conducted a 24-hour aerobic incubation using the same jars to mimic what

would occur in the natural environment during a period with a lower water table. We opened the

sealed jars and ran a high-powered fan over top for 10 minutes to flush the jar headspace with

room air. After the aeration, we sealed the jars again and put them back in the growth chamber

under dark conditions at 15 °C. This time when sampling the gas, we filled the syringes with

room air prior to sampling, rather than N2 gas. We sampled the headspace gas from each jar one

hour after the aeration, and after 12 and 24 hours. We measured for CO2 using the same

instrument, method quality control procedure and calculations as above. After this incubation,

we oven-dried the peat at 70 °C for 48 hours and weighed it. We then expressed the CO2 and

CH4 mass on a dry mass basis.

We analyzed for responses in 40-day anaerobic CO2 and CH4 and 24-hour aerobic CO2,

using StatisticaÒ version 10 and a 5% type I error rate. We log-transformed all the data. We first

analyzed for species effects using one-way analyses of variance. We then conducted factorial

analyses of variance separately by Sphagnum species to analyze for significant effects of peat

origin and nutrient amendment and their interaction. We used post-hoc Tukey tests to further

examine significant effects.

Results

Sphagnum species had significantly different CO2 respiration after 40 days under anaerobic

conditions, across all peat origins and nutrient amendments (P < 0.0001; Figure 1a). S. fuscum

had the lowest anaerobic respiration of CO2 (mean:7000 µg g dry mass-1), and it differed

54

significantly from both S. rubellum and S. majus (both: Tukey P < 0.0001), but S. rubellum and

S. majus were not significantly different from each other (Tukey P = 0.276; S. rubellum mean:

9930 µg g dry mass-1; S. majus mean:11190 µg g dry mass-1). The CO2 respiration of S. fuscum

peat after 40 days of anaerobic conditions strongly depended on the origin of the peat

(P = 0.0002; Table 1); the peat from the start of the experimental fen had significantly greater

respiration than the peat from both the end of the experimental fen and the reference fen, which

were not different from each other (Figure 1a). However, nutrient amendment had no significant

effect on the anaerobic microbial respiration of S. fuscum peat, nor was there any interaction with

peat origin (Figure 2a). We found a similar pattern for S. rubellum. Its anaerobic respiration also

depended on the origin of the peat (P = 0.0002), again with more CO2 production for peat

originating from the start of the experimental fen than from either the end of experimental fen or

reference fen. Nutrient amendment again had no effect on the anaerobic CO2 respiration of S.

rubellum peat, again with no interaction. In contrast to the other species, the anaerobic CO2

respiration of S. majus peat was not affected by its origin nor by any nutrient amendment.

When we examined the subsequent aerobic CO2 respiration from the remnant peats over

24 hours, it was only half an order of magnitude less across all species than the anaerobic CO2

respiration over 40 days (Figure 1b). All three Sphagnum species now strongly differed from

each other (all species: Tukey P < 0.0001), with S. fuscum having the lowest 24-hour CO2

aerobic respiration rate (mean: 1670 µg g-1 dry mass), followed by S. rubellum (mean: 2700 µg

g-1 dry mass), and S. majus (mean: 3400 µg g-1 dry mass). The aerobic CO2 respiration from

S. fuscum peat did not depend on the origin of the peat (Table 1), but it did depend on the level of

nutrient amendment (P < 0.0001; Figure 2b), and there was a significant peat origin by nutrient

interaction. Peat originating from the start and end of the experimental fen with 10x field-loading

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concentration of nutrients, produced a significantly lower amount of CO2 that the 0x, 0.5x, 1x, or

2x field-loading nutrient concentrations, whereas the reference fen peat samples produced similar

amounts of CO2 across all nutrient amendment levels (Figure 3).

Aerobic CO2 respiration of S. rubellum peat strongly differed depending on the peat origin

(P < 0.0001; Table 1, Figure 1b); peat from the start of the experimental fen produced the

greatest CO2, while peat from the end of the experimental fen produced the least, and the

reference fen produced intermediate levels. However, nutrient amendment had no significant

effect on the aerobic CO2 respiration of S. rubellum peat, nor was there any interaction with peat

origin. S. majus behaved similarly. Aerobic CO2 respiration of S. majus peat strongly depended

on its origin (P < 0.0001; Table 1; Figure 1b), with peat from the start of the experimental fen

producing the greatest CO2, peat from end of the experimental fen producing the least, and the

reference fen having intermediate levels (Figure 1b). Nutrient amendment had no significant

nutrient effect on the aerobic CO2 respiration of S. majus peat, nor was there any interaction with

peat origin (Table 1).

When we examined the anaerobic CH4 production after the 40-day incubation, it was

between two and seven orders of magnitude lower than anaerobic CO2 production (Figure 1c).

All three species of Sphagnum were significantly different. S. fuscum produced extremely low

amounts of CH4 (mean: 0.21 µg g-1 dry mass), while S. rubellum produced an intermediate

amount (mean: 1.33 µg g-1 dry mass), and S. majus produced the greatest CH4 (mean: 21 µg g-1

dry mass). The CH4 production from S. fuscum peat did not depend on its origin, but it did

depend on the nutrient amendment (P = 0.010; Table 1). CH4 production was lowest under the 0x

nutrient treatment, and increased to a peak at the 2x nutrient treatment, then CH4 production

declined when 10x nutrients were applied (Figure 2c). In contrast, the CH4 production of S.

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rubellum peat strongly depended on the origin of the peat (P < 0.0001); peat from the start of the

experimental fen produced the most CH4, while peat from the end of the experimental fen

produced the least, and peat from the reference fen was intermediate (Figure 1c). Nutrient

amendment also caused a difference in CH4 production for S. rubellum peat (P = 0.002), but with

a significant interaction with the peat origin (P = 0.011). CH4 production dropped especially at

the 10x nutrient amendment (Figure 2c), but this was especially evident for peat from the start of

the experimental fen and did not occur at the end of the experimental fen (Figure 4). Finally, the

CH4 production from S. majus peat depended on the peat origin (P = 0.0002); the start of the

experimental fen produced similar amounts of CH4 as the reference fen, while the end of the

experimental fen produced less CH4 (Figure 1c). Nutrient amendment had no effect on CH4

production from S. majus peat (Figure 2c) and no interaction occurred.

Discussion

We examined Sphagnum decomposability across three different Sphagnum species, collected

from origins that differed in prior productivity, and along a gradient of nutrient amendments. We

aimed to predict how Sphagnum-peat decomposition (mineralization of carbon) may change

throughout the HBL peatlands should they become used to polish nutrients present within treated

domestic wastewater.

We had first hypothesized that a hummock-forming species (Sphagnum fuscum) would

decay more slowly than a carpet species (Sphagnum rubellum), followed by a hollow species

(Sphagnum majus). Our results support our first hypothesis. S. fuscum produced the least CO2

and CH4, S. rubellum produced intermediate amounts, and S. majus produced the greatest,

showing the most decay potential. Hummock forming species, such as S. fuscum, contain a

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greater concentration by mass of highly recalcitrant complex organic compounds (polyphenolic

compounds and uronic acids) than hollow species (Clymo 1963; Kälviäinen and Karunen 1984;

Johnson and Damman 1991; Turetsky et al. 2008). In our results, the hollow>carpet>hummock

decay pattern held true under anaerobic and aerobic conditions regardless of which location of

peat origin or level of nutrient amendment. Our results support the conclusions from previous

research that determined that variations in microbial decomposition across different Sphagnum

species is primarily due to their variations in litter chemistry, specifically percentage of highly

recalcitrant phenolic compounds (Berendse et al., 2001; Bragazza et al., 2005).

We also had hypothesized that, following two seasons of high nutrient loading, peat

produced under higher nutrients, originating from the start of the experimental fen, would decay

most rapidly, producing greater amounts of CO2 and CH4 than peat originating from the end of

the experimental fen and a reference fen. Generally, we found that microbial CO2 respiration and

CH4 production levels strongly differed by peat origin, supporting our second hypothesis. In

most cases throughout this experiment, peat originating from the start of the experimental fen

produced the most CO2 and CH4 outputs, showing greater decomposability compared to peat

originating from the end of the experimental fen or reference fen. This result suggests that high

productivity Sphagnum peat grown in locations exposed to a higher nutrient loading decompose

more than low productivity Sphagnum peat, and supports the findings from other peatland

fertilization studies (Aerts et al., 2001; Bubier et al., 2007). Increasing the N content within

Sphagnum peat and lowering the C:N ratio leads to faster rates of decomposition (Coulson and

Butterfield, 1978; Clymo and Hayward, 1982). Nutrient content results from our previous study

(Chapter 1) confirm that all three species had significantly lower C:N values in the peat samples

collected from the start of the experimental fen location. S. fuscum had 1.5x lower C:N values, S.

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rubellum 3x lower, and S. majus samples had 3.5x lower C:N values at the start of the

experimental fen compared to the end of experimental fen or reference fen levels (Chapter 1),

showing interspecific variation in nutrient retention across these origins.

However, some results from this study were exceptions to the general trend with peat

origin, specifically, S. majus CO2 production under anaerobic incubation, and S. fuscum aerobic

CO2 and anaerobic CH4 production, which showed no significant difference across peat origin

(Table 1; Figure 1). For these exceptions, we can suggest that the intraspecific litter nutrient

content within the peat from all three origins may not have been significantly different from one

another. In the case of S. majus CO2 production under anaerobic conditions, we suspect that the

S. majus surface peat tissues collected from the start of the experimental fen were primarily

composed of newer growth (younger) tissues that grew quickly, and may not have had time to

accumulate (absorb) the field-loaded nutrients and therefore, N, P, K, and S concentration within

those samples may have been similar to those from the non-fertilized older S. majus tissues

found at the end of the experimental fen and reference fen.

Similarly, for S. fuscum aerobic CO2 and anaerobic CH4 production, we believe that while

S. fuscum was in the field during the field-loading of treated wastewater nutrients experiment

(2015 growing season), the fertilized water table stayed between 10 – 40 cm below the top

surface of the high hummocks on the peat ridges where S. fuscum grows (McCarter, 2016),

therefore, the start of the experimental fen S. fuscum tissues could have had comparable nutrient

concentrations to the end of the experimental fen and reference fen tissues.

Interestingly, our experimental results did not support our third hypothesis proposing that

with greater nutrient amendment levels would yield greater CO2 and CH4. We found that under

anaerobic and aerobic incubation, the addition of nutrients to the incubation jars did not

59

significantly increase CO2 or CH4 production, and thus did not increase decay potentials. Similar

studies within the literature contain mixed results regarding the effects of nutrient additions on

peat decay. Some peat incubation studies found that treatments with N, and N and P, lead to

significantly greater decomposition (Coulson and Butterfield 1978; Hogg et al. 1994; Aerts and

Toet 1997). However, many other studies had results similar to this study where nutrient

additions had no significant increase to microbial CO2 and CH4 emissions (Bartsch and Moore

1985; Williams and Silcock 1997; Hoosbeek et al. 2002). Amador and Jones (1993) and Aerts et

al. (2001) found no increase in peat CO2 respiration with increased P fertilization, however, in

more nutrient poor ombrotrophic peatlands, both N and P are predicted to be limiting decay and

P additions significantly increase CO2 production (Aerts et al. 1992; Hogg et al. 1994).

Many past peatland fertilization experiments have nutrient fertilization rates at much

lower doses than this study, making comparison of our results to theirs difficult. For example, the

ombrotrophic peatland outside Ottawa Ontario (Mer Bleau bog) received N loading treatment

ranging from 1.6 – 6.4 g N m-2 year-1 (Bubier et al., 2007), and our field-loading N application

level (1x nutrient amendment treatment) would be approximately 9.4 – 15.3 g N m-2 year-1, and

our 10x nutrient loading amendment level would equate to 94 – 153 g N m-2 year-1.

Limpens and Berendse (2003), suggest that within some of the peatland fertilization

studies where results showed no increase to decay, that the cause could be that the C:N ratio of

the Sphagnum litter may not have decreased enough to see a positive effect on decay potentials.

If this theory applied here to our study, we would suspect that Sphagnum tissues from the start of

the experimental fen with 10x nutrient loading would have low C:N ratios and increase their

decay potentials, yet we did not see this result. In fact, we noted a significant decrease in CO2

emission from the S. fuscum, and a decrease in CH4 emissions from S. rubellum at the highest

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(10x) nutrient loaded level (Figure 2). This result implies a theoretical threshold where above

that level of nutrient loading, microbial decay declines or is inhibited, and furthermore, these

thresholds may differ depending on the Sphagnum species.

The theory of optimal ranges of nutrient ratios, (C:N, C:P, C:K, C:S), or critical ratios

within Sphagnum peat has been mentioned as explanation to the variability of results within the

literature regarding nutrient fertilization effects on Sphagnum decomposition (Moore et al.,

2011; Wang et al., 2014). Lamers et al. (2000) and Berendse et al. (2001) suggest that low levels

of N deposition, Sphagnum quickly absorb the added N, but under increasing input Sphagnum

become N-saturated to the point where they lose their N filtering capacity. Bragazza et al.,

(2005) added to this theory by conforming that within regions with higher N deposition, the

decreasing retention of N within the Sphagnum was accompanied by an exponential increase in

the concentration of inorganic N in the surrounding pore water.

Perhaps a similar principle may apply for other nutrient ratios such as the C:P, C:K, and

C:S ratios within peat where there is an optimal range where microbial metabolization occurs in

greater amounts, and additions above that range may be toxic to microbes and could cause a

decrease in respiration, thus decreasing their decay potential (Wang et al., 2014). Similarly, the

10x nutrient loading amendment may have contained enough ammonia (NH4) to be toxic for the

microbes, causing an inhibitory effect to microbial respiration. Waste water contains high

concentrations of NH4, and high concentrations have been determined to decrease microbial

activity (Lee et al., 2000). Anthonisen et al., (1976) found that 10 – 150 mg L-1 of NH4 was

inhibitory to many heterotrophic microbes. The 10x nutrient amendment within this experiment

contained 91 mg L-1 of NH4, which falls within the identified rage of NH4 toxicity, and therefore,

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could be the cause for significantly less microbial respiration from the highest nutrient

amendment level.

We know that Sphagnum species can vary in litter chemistry, specifically their carbon:

polyphenol compound ratios (Kälviäinen and Karunen 1984; Turetsky et al. 2008). Bragazza et

al. (2006), found that the C:N ratio within Sphagnum is strongly positively correlated with

polyphenol concentrations, and the C:N ratio also positively correlates with other nutrient ratios

such as C:P and C:K, and therefore, the C:N ratio is an excellent predictor of Sphagnum

decomposability, and supports that variations to litter chemistry and nutrient content translates to

changes in decay potentials.

In this study, S. fuscum decay was significantly lower at 10x nutrient loading levels. We

suspect that the microbes may have become substrate limited as the amount of easily labile

organic carbon may have been previously depleted. The remaining organic carbon was likely

bound within highly recalcitrant polyphenolic compounds and unavailable to most microbes for

easy mineralization. S. fuscum is a slow growing species, thus the ~10 cm of surface peat

collected for this study would have been older in age and had a greater concentration of

polyphenols relative to the faster growing (newer growth) of the S. rubellum and S. majus

samples, particularly within the start of experimental fen location where growth rates were

significantly faster than the end of experimental fen or reference fen (Chapter 2). Therefore, the

differences in decomposition could be due to differences in chemical aging of the peat substrate,

which was also the conclusion from a similar Sphagnum peat decomposition study (Belyea,

1996).

However, the pattern of increased nutrients causing decreased microbial respiration was

location specific as it only occurred within the two experimental fen locations, and not within the

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reference fen. Reference fen S. fuscum peat produced similar CO2 outputs across all nutrient

treatment levels (Figure 3). This result lead us to suspect that perhaps there were abiotic

environmental factors (temperature, pH, or water level) that differed between the two ribbed fens

sites allowing for some variations to either litter structure or litter chemistry, and allowed for

more tolerance to higher levels of nutrient loading.

An environmental factor within the incubation environment that may have been altered

with high nutrient loading is pH. Perhaps the 10x nutrient loading level caused a decrease to pH

to the point where microbial respiration and Sphagnum decay was inhibited. Greater additions of

NO3, NH4, PO4, and SO4 nutrients can increase soil acidity (Parchomchuk et al., 1993). Preston

et al., (2012) concluded that pH was a strong predictor of microbial activity within peatland

soils. Other studies have concluded that microbial diversity and activity is greater at more neutral

pH levels (6-7) rather than acidic pH levels (3-5) (Fierer and Jackson, 2006; Rousk et al., 2010).

A limitation of our study was that we did not measure the pH of the pore water within the

incubation jars throughout our experiment, therefore, we are unable to determine if there was a

significant pH decrease within the 10x nutrient loading treatments, that may be contributing to

the decrease in CO2 outputs from S. fuscum and S. majus at the 10x nutrient amendment level.

Upon assessing CH4 production across our nutrient amendment rates, we found that S.

fuscum and S. rubellum produced the least amount of CH4 when fertilized at the highest

treatment level (10x) (Figure 2). This result also leads us to suspect that high nutrient loading

may inhibit some pathways within the process of Sphagnum decay, and that the amount of

nutrient amendment required to see this inhibition, differs between Sphagnum species, as S.

majus produced a similar amount of CH4 across all amendment levels. In this case, we predict

that the high level of sulfate (SO4-2) within our simulated wastewater fertilizer is likely the cause

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for the decrease in CH4 production for S. fuscum and S. rubellum. S has been identified as

playing an important role in the oxidation-reduction pathway within peatlands (Gauci et al. 2002;

Gauci et al., 2004), and as a result, additions of SO4-2 have been linked to a reduction or

inhibition of CH4 emissions (Kristjansson et al., 1982; Yavitt et al., 1987; Blodau and Moore,

2003). Briefly, under anaerobic conditions, when SO4-2 is increased in availability, the dominant

terminal respiration process will become more favorable for sulfate reducing bacteria rather than

methanogens (methane producing bacteria) because SO4-3 reduction requires less energy

(Kristjansson et al., 1982; Yavitt et al., 1987). We presume that S. majus could sustain a

consistent output of CH4 across all nutrient amendment levels because it develops under

anaerobic water saturated conditions (Clymo and Hayward 1982), which are also the conditions

required for methanogens (Dunnfield et al., 1993) and thus as a species may have developed

more favorable litter structure and chemical composition for methanogens in comparison to S.

fuscum and S. rubellum. Furthermore, microbial CH4 production is suspected to be more

sensitive to environmental changes, for example temperature, pH, water level, and redox-

oxidation potential than CO2 production (Moore and Dalva, 1993; Lai, 2009). While the peat was

within its incubation environment in the lab, we could control temperature and water level,

however, prior to peat harvest while the moss was developing and under earlier stages of

decomposition, these environmental factors were highly variable within the natural field

conditions, which may account for variability of litter structure within a species or across peat

origin.

Overall, Sphagnum decay potentials significantly differed across hummock, carpet, and

hollow species, and across peat origins, however, we suspect that the key factor that varied

between these three species and these three peat origins is their variations in litter chemistry. In

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addition, because peat origin within this experiment was highly significant, we believe that

nutrient uptake within the Sphagnum tissues while they are still active (photosynthetic) has a

greater effect on the decay potential of the Sphagnum once it becomes litter than does adding

additional nutrient amendments to the incubation environment once the Sphagnum-peat is

already inactive (non-photosynthetic). Therefore, long-term in-situ (field-based) experiments

studying the pattern of CO2 and CH4 emission from these three species in their respective

microhabitats would be valuable to better understand their evolution of decay within their natural

environments from the point of productivity to the point they begin to senesce.

Understanding how nutrient additions can change the decomposability of Sphagnum peat is

critical for predicting further changes to the physiological structure of the peat within these HBL

ribbed fens. Ribbed fens are desirable systems for wastewater polishing because of their pattern

of raised peat ridges (hummocks) interspersed with hollow pools where the ridges impede

hydrological flow and the nutrient deprived Sphagnum scavenge the added nutrients

immobilizing it from the pore water further downstream (McCarter, 2016). Increases or

decreases to the rate of decay within these peat ridges could change the hydrological properties

of these mires, possibly rendering them less effective at polishing wastewater. Additional long-

term research on how high hydrological loading and nutrient loading would change the

physiological structure of these ribbed fens would be beneficial for providing further comment

on the feasibility of these peatlands to continue to polish wastewater long term.

The number one ecosystem service provided by these northern HBL peatlands is their

ability to store carbon because their net uptake of atmospheric CO2 far exceeds their release of

CO2 or CH4 back into the atmosphere. For these systems to remain a globally significant carbon

sink, their rate of primary productivity must remain greater than their rate of decay. Our short-

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term field study (Chapter 1) concluded that with point source nutrient application, productivity

remained greater than decomposition. Within this short-term incubation study, higher nutrient

loading levels did not significantly increase the rate of CO2 or CH4 emissions, which again

suggests that these Sphagnum species are tolerant of high nutrient loadings.

Other long-term peatland fertilization experiments concluded that Sphagnum decay rates

increase with increasing nutrient content (Clymo 1965; Heal et al., 1978), and Sphagnum decline

in abundance as they become out competed by other vascular plants, such as grasses and shrubs

(Bubier et al., 2007; Bragazza et al., 2004; Berendse et al., 2001). As grasses, sedges, and shrubs

have faster rates of decomposition relative to Sphagnum (Rydin and Jeglum 2013), if these plant

community shifts were to occur within the HBL peatlands, than long-term net carbon storage of

these systems may diminished. Further field research on plant community shifts within the HBL

peatlands experiencing higher nutrient loading would be beneficial.

Conclusion

The HBL peatlands are under increased pressure for mining development and anthropogenic

influences on the landscape. Should the population of the far north Ontario increase, the need for

wastewater polishing will also increase. Few studies have been conducted on the effects of

adding point source nutrients that contain nutrients similar to what would be present within

treated wastewater directly to a ribbed fen within a northern subarctic climate. We found that

peat nutrient content (concentration of organic bound C, N, and P) within peat plays a large role

in the decay potential for Sphagnum-based peat, and the optimal nutrient ratios within the litter

likely differs at the species level. Highly recalcitrant hummock forming Sphagnum species are

more resistant to decomposition than carpet and hollow species, however hummock species may

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be more likely to leech labile inorganic nutrients while under high nutrient loading. We provide

evidence that litter chemistry and nutrient uptake (and retention) within living Sphagnum has a

greater influence on the decomposability of that material than fertilization to non-living

Sphagnum peat. Our results support findings that the litter chemistry variables such as

polyphenols: nutrient ratios, and C: nutrient ratios appear to be the primary variables controlling

microbial decomposition of Sphagnum moss (Limpens and Berendse, 2003; Bragazza et al.,

2006), and carbon mineralization rates (Yavitt et al., 1987).

Acknowledgements

The research was funded through the NSERC Canadian Network for Aquatic Ecosystem

Services (CNAES) and De Beers Canada. The first author gratefully acknowledges the salary

support through the NSERC Industrial Postgraduate Scholarship program. We are also grateful

for travel support provided by the Northern Scientific Training Program.

We would like to acknowledge the help of committee members Dr. Nathan Basiliko and

Dr. Graeme Spiers for their constructive criticism. We thank field assistants Angela Borynec and

Ainsley Davison, as well as graduate students Andrea Hanson and Brittany Rantala-Sykes, and

network collaborators Drs. Colin McCarter, Jonathan Price, and Brian Branfireun. We sincerely

thank all employees at the De Beers Victor Mine who assisted us along the way, especially

Stephen Monninger, Terry Ternes, Anne Bouche, Rod Blake, Nick Gagnon, Jake Carter, and all

members of the Environment and Reclamation department.

Thank you to fellow Vale Living with Lakes graduate students and professors whom have

either provided me with training in the lab, advice on data analysis, or allowed me to use their

67

equipment, specifically Michael Carson, Gretchen Lescord, Dr. Tom Johnston, and Dr. Nathan

Basiliko.

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Table 1. Separate analyses of variance by Sphagnum species of 40 day anaerobic CO2 release, 24 hour aerobic CO2 release and 40 day

anaerobic CH4 release during the incubation experiment as a function of Sphagnum origin, nutrient amendment and their interaction.

Analyses were based on log-transformed data. Results with type I error level < 5% are shown in bold.

40dayanaerobicCO2 24hraerobicCO2 40dayanaerobicCH4Species Sourceofvariation df MS F P MS F P MS F P

Sphagnumfuscum origin 2 0.238 11.5 0.0002 0.021 2.6 0.088 0.206 2.7 0.083

nutrients 4 0.005 0.2 0.907 0.074 9.4 <0.0001 0.305 4.0 0.010

originxnutrients 8 0.018 0.9 0.552 0.027 3.4 0.006 0.057 0.7 0.650

error 30 0.021 0.008 0.076 Sphagumrubellum origin 2 0.233 12.0 0.0002 0.815 88.5 <0.0001 19.113 115.0 <0.0001

nutrients 4 0.021 1.1 0.393 0.015 1.6 0.189 0.877 5.3 0.002

originxnutrients 8 0.009 0.4 0.885 0.020 2.2 0.058 0.515 3.1 0.011

error 30 0.020 0.009 0.166 Sphagnummajus origin 2 0.012 0.6 0.577 0.449 25.9 <0.0001 9.453 12.0 0.0002

nutrients 4 0.030 1.4 0.271 0.038 2.2 0.092 0.770 1.0 0.436

originxnutrients 8 0.020 0.9 0.522 0.018 1.1 0.418 0.251 0.3 0.953 error 30 0.022 0.017 0.790

74


production, and (C) the 40 day anaerobic CH4 production during the incubation of peat from

Sphagnum fuscum (brown), S. rubellum (red) and S. majus (green) collected at the start and the

end of the experimental fen and at the reference fen, across all nutrient amendment treatments.

Letters above box plots show results of post-hoc Tukey test with a 5%Type I error rate, plots

with no letters indicates no significant differences across peat origin.

3000

10000

30000

600

1000

10000

0.01

0.1

1

10

100

1000

StartEXP

EndEXP

REF StartEXP

EndEXP

REF StartEXP

EndEXP

REF

S. fuscum S. rubellum S. majus

40 d

ay a

naer

obic

CO

2 (μ

g g-

1 dr

y m

ass)

24 h

r aer

obic

CO

2 (μ

g g-

1 dr

y m

ass)

40 d

ay a

naer

obic

CH

4 (μ

g g-

1 dr

y m

ass)

(A)

(B)

(C)

a b b

a b b

a b ba b b

a ab b

a b cba a

75


production, and (C) the 40 day anaerobic CH4 production during the incubation of peat from

Sphagnum fuscum (brown), S. rubellum (red) and S. majus (green) as a function of nutrient

amendment levels ranging from zero (no amendment) to up to 10x the field rate of nutrient

amendment. Letters above box plots show results of post-hoc Tukey test with a 5%Type I error

rate, plots with no letters indicates no significant differences across nutrient amendment.

0 1020.5 1 0 1020.5 1 0 1020.5 1

S. fuscum S. rubellum S. majus

24 h

r aer

obic

CO

2 (μ

g g-

1 dr

y m

ass)

Nutrient amendment

40 d

ay a

naer

obic

CH

4 (μ

g g-

1 dr

y m

ass)

600

1000

10000

0.01

0.1

1

10

100

1000

3000

10000

30000

40 d

ay a

naer

obic

CO

2 (μ

g g-

1 dr

y m

ass)

(A)

(B)

(C)

a a a a b

a a a ab b

a ab ab abb

76

Figure 3. Box plots of the CO2 produced after 24 hours under aerobic conditions for S. fuscum at

each nutrient amendment level, ranging from zero (no amendment) to up to 10x the field rate of

nutrient amendment, at all three peat origins. Letters above box plots refer to similar groups

based on post hoc Tukey test with a 5%Type I error rate.

start EXP end EXP REF

600

1000

6000

0 0.5 1 2 10 0 0.5 1 2 10 0 0.5 1 2 10

24 h

r aer

obic

CO

2(μ

g g-

1 dr

y m

ass)

Nutrient amendment

a a a b ba a a a a a a a a aba

77

Figure 4. Box plots of the CH4 produced after 40 days under anaerobic conditions for S.

rubellum at each nutrient amendment level, ranging from zero (no amendment) to up to 10x the

field rate of nutrient amendment, at all three peat origins. Letters above box plots refer to similar

groups based on post hoc Tukey test with a 5%Type I error rate.

start EXP end EXP REF

40 d

ay a

naer

obic

CH

4(μ

g g-

1 dr

y m

ass)

0 0.5 1 2 10 0 0.5 1 2 10 0 0.5 1 2 10

Nutrient amendment

0.01

0.1

1

10

100

1000a a a a

b b b b b b b b b b b

78

Research Implications Sphagnum is the keystone genus within northern subarctic peatlands. Its health is closely tied to

the quality and quantity of ecosystem services that these peatlands can provide. Our field-based

research (chapter 1) found that short-term additions of simulated secondarily-treated wastewater

to an experimental ribbed fen increased the primary productivity of Sphagnum, while decay rates

remained low and comparable to an unamended ribbed reference fen. This implies that

wastewater polishing will produce greater peat formation and greater carbon sequestration over

the short term in these ribbed fens. Our incubation study (chapter 2), found that changes in

quality and nutrient content of Sphagnum litter brought on during wastewater polishing play a

larger role in its decay potential than the addition of further nutrients during the decomposition

process. Ultimately, Sphagnum litter chemistry variables, possibly polyphenols: nutrient ratios,

and C: nutrient ratios, appear to be the primary variables controlling microbial decomposition of

Sphagnum moss.

Changes Sphagnum productivity and decomposition found in this study must be considered

in light of the in hydrology, nutrient transport and geochemistry. McCarter et al. (2017)

determined that the experimental ribbed fen at Victor Mine was highly effective at removing the

added nutrients from the pore water as none of the added nutrient contaminants were detected at

the outflow of the fen. The nutrients most common to domestic wastewater (NO3, NH4, and PO4)

remained effectively immobilized within the first or second peat ridge, and the SO42- plume,

although transported further downgradient than the other nutrient contaminants, was still

effectively polished from the pore water, proving that the ribbed fen peatland hydrology and

structure can be successful systems for wastewater polishing (McCarter et al., 2017).

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Further research on the biogeochemical interactions within the experimental fen at Victor

Mine is ongoing especially in regard to methyl mercury (MeHg; L. Twible, Western University,

in preparation). The ground water within the HBL region is rich in SO42-, causing the domestic

wastewater produced in the region to also be elevated in SO42- concentrations (Steinback, 2012).

Added SO42- to waterlogged anoxic peat has been found elsewhere to lead to increased levels of

MeHg, as the sulphate-reducing bacteria can facilitate methylation of mercury under these

anaerobic conditions (Branfireun et al., 1999; Mitchell et al., 2008). Methyl mercury is a

biological toxin, and once in the aquatic food web can bioaccumulate and biomagnify (Kidd et

al., 1995). As this research on MeHg biogeochemistrybecomes available, it can be integrated

with the hydrological results (McCarter and Price 2017; McCarter et al., 2017) and Sphagnum

growth and decomposition results (this study) to reach broader conclusions on the overall

benefits of using ribbed fens to polish secondarily-treated wastewater.

Certainly, similar ribbed fens exist across boreal and subarctic Canada in the shield and in

the HBL (Zoltai et al., 1988; Riley, 2011). We only looked at one treatment ribbed fen,

compared to one reference fen, but the studied fens appear to fairly represent other ribbed fens in

the subarctic, in terms of their morphology and composition, if not in size. Given this similarity,

we do not see how our Sphagnum productivity and decomposition results would not apply over

the short term to other ribbed fens, if the nutrient and water inputs were scaled to the size of

ribbed fens. Further study would be required to compare the hydrology and Sphagnum

communities across a series of ribbed fens.

The experimental ribbed fen wetland at De Beers Victor Mine received simulated

secondarily-treated domestic wastewater for two consecutive growing seasons (summer 2015

and 2016). We do not know what the medium or long term effects would be on Sphagnum

80

growth or productivity if fertilization were to continue. In theory, the additions of N and P to the

peatland system would allow for vascular plants to obtain required nutrients to survive and grow.

They will be able to produce taller shoots, thereby creating above-ground competition for light,

out-shading the smaller plants (Moore et al., 1989; Wisheu and Keddy, 1992), including

Sphagnum mosses. The taller plants will also produce more litter which will allow a secondary

competitive effect over ground-dwelling plants (Facelli et al., 1991), such as Sphagnum mosses.

Kadlec and Bevis (2009), found a strong shift to a tall and dense Typha dominated community

after 30 years of nutrient addition in a more southern peatland which received secondarily treated

wastewater. Based on these results from previous research, we predict that with longer-term

nutrient addition, the plant community will shift away from Sphagnum dominance and move

towards taller species such as tall graminoids and shrubs (Kadlec and Bevis, 2009; Bubier et al.,

2007; Bragazza et al., 2004; Berendse et al., 2001). If vascular plants eventually out-shade the

Sphagnum, any positive affect of increased Sphagnum productivity and increased peat formation

would be temporary. The length of time that these subarctic ribbed fens can uptake nutrient

without experiencing significant reduction to Sphagnum cover because of competition with taller

vascular plants remains unknown.

To test this prediction of longer term effects of wastewater polishing in ribbed fens, the

treatment fen would have to be continued, perhaps for as much as a decade. The establishment of

many permanent vegetation sampling plots prior to any nutrient additions would also have been

necessary. Only a few sampling plots were set up prior to the experiment, insufficient for any

examination of changes in community assemblages over the short term. We can only comment

on our visual observations of plant cover and diversity in summer 2015 verses summer 2016.

81

Both Dr. McCarter and I observe that the cover of graminoids appears as though it had increased

within the first 50 meters down gradient from the point source nutrient load.

Again, longer-term hydrological and geochemical changes are also unclear. Kadlec (2009)

found large shifts in the hydrology and nutrient relations of their treatment peatland over its 30-

year lifespan. Further research would also be required on these aspects in subarctic ribbed fens.

The recovery time of these fens after the cessation of nutrient amendment is also unclear.

Few studies have studied the addition of secondarily treated wastewater to peatlands. We know

of no studies that examined the impacts on plant communities or ecosystem function after these

waste water additions have been halted.

If all the short-term effects are sufficiently benign, one option to limit any (unknown)

longer-term effects would be to move the outflow pipe into new polishing ribbed fens every few

years. There are many unknowns with such a scenario, including the prospect of larger scale

cumulative effects, requiring careful study, monitoring and management. Ultimately, the

precautionary principle should apply.

There has been increasing pressure for industrial development on Ontario’s far north (Far

North Science Advisory Panel, 2010). If additional mines were to operate within the Hudson Bay

Lowland, development planners will be required to have an environmentally responsible method

for treating then polishing their domestic wastewater. The use of a natural wetland system to

polish secondarily-treated wastewater, if selected carefully and well managed, could be more

cost-effective relative to engineered tertiary treatment facilities. Ontario’s far north is highly

remote, transporting the good and materials needed for infrastructure is difficult, therefore

developers may seek to reduce the amount of specialized infrastructure required for operation.

82

However, given the unknowns on the longer-term function of treatment ribbed fens, it is difficult

to recommend the broader use of polishing peatlands, at least without further research.

This thesis suggests that the Sphagnum dominant nutrient-poor peatland within the HBL can

successfully polish treated domestic wastewater over the short term, but there are still many

uncertainties, some over the short term and many over the longer term. Research should be

completed on a full range of potential environmental impacts associated with northern treatment

wetlands and or polishing wetlands prior to recommending wider use of ribbed fens for

wastewater polishing within the HBL. Until this research becomes available, it is difficult to

consider in detail the implications, benefits, or impacts associated with the use of natural

peatlands to polish wastewater in the far north.

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Appendix

Figure A1. Scatter plot of Total Nitrogen content (percent mass) within Sphagnum fuscum (top),

Sphagnum reubellum (centre), and Sphagnum majus (bottom) over distance (m), experimental

fen (solid black circles) and the reference fen (open circles). The vertical dashed line marks the

location of the discharge point of nutrients, and negative distance values correspond to sample

sites up gradient from the point source.

Distance (m)

Tota

l Nitr

ogen

(%)

S. fuscum

S. rubellum

S. majus

85

Figure 2A. Scatter plot of Total Sulfer content (percent mass) within Sphagnum fuscum (top),





Tota

l Sul

fer (

%)

Distance (m)

S. fuscum

S. rubellum

S. majus

86

Figure 3A. Scatter plot of Phosphorus concentration (mg kg-1) within Sphagnum fuscum (top),





S. fuscum

S. rubellum

S. majus

Distance (m)

Phos

phor

us (m

g kg

)

-1

87

Figure 4A. Scatter plot of Potassium concentration (mg kg-1) within Sphagnum fuscum (top),





Pota

ssiu

m (m

g kg

)

-1

Distance (m)

S. fuscum

S. rubellum

S. majus

88

Figure 5A. Scatter plot of Calcium concentration (mg kg-1) within Sphagnum fuscum (top),





Cal

cium

(mg

kg

) -1

Distance (m)

S. fuscum

S. rubellum

S. majus

89

Figure 6A. Scatter plot of Magnesium concentration (mg kg-1) within Sphagnum fuscum (top),





Mag

nesi

um (m

g kg

)

-1

Distance (m)

S. fuscum

S. rubellum

S. majus

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